Research Of ⁶⁸Ga-labeled Radiotracers ⁶⁸Ga-DOTA-TMTP1 And⁶⁸Ga-NOTA-?TMVP1?₂ In Tumor-targeted Molecular Imaging

PurposeMolecular imaging enables visualization and characterization of biological processes that influence tumor behavior and response to therapy.TMTP1?NVVRQ?is a novel peptide previously identified by our laboratory and has shown remarkable affinity to highly metastatic tumors and occult metastasis foci.In this study,we have designed and synthesized a ⁶⁸Ga-labeled cyclic TMTP1 radiotracer,⁶⁸Ga-DOTA-TMTP1,for PET imaging of cervical cancer.The goal of this study was to investigate the properties of this radiotracer and its tumor diagnostic potential.MethodsDOTA-TMTP1 was synthesized by Fmoc solid-phase peptide synthesis and labeled with ⁶⁸Ga by manual method or semi-automated module.The product was purified by Sep-pak C18 Light solid-phase extraction column and quality control of the product was performed.Theoctanol-waterpartitioncoefficientandthestabilityof⁶⁸Ga-DOTA-TMTP1 in vitro were determined.The cell uptake and efflux of⁶⁸Ga-DOTA-TMTP1 was examined in paired highly metastatic and lowly metastatic cervical cancer cell line Hela and C-33A as well as normal cervical epithelial cell line End1.The cell uptake blocking experiment was conducted to confirm the specific binding of ⁶⁸Ga-DOTA-TMTP1.1h-dynamic PET scans were performed using normal mice to determine in vivo biodistribution and pharmacokinetics of ⁶⁸Ga-DOTA-TMTP1.Static PET images were acquired at different time points post injection using the Hela and C-33A tumor-bearing mice.The in vivo blocking assay and biodistribution study were performed in Hela tumor-bearing mice.ResultsTMTP1 was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid?DOTA?and successfully labeled with ⁶⁸Ga.The radiochemical yield of⁶⁸Ga-DOTA-TMTP1 was high and the radiochemical purity was greater than 95%.The tracer was found to be safe and non-toxic.The octanol-water partition coefficient for⁶⁸Ga-DOTA-TMTP1 was-2.76±0.08 and ⁶⁸Ga-DOTA-TMTP1 has showed excellent stability in in vitro studies.⁶⁸Ga-DOTA-TMTP1 exhibited higher uptake in Hela cells than in C-33A cells.The binding to Hela and C-33A cells could be blocked by excess TMTP1.⁶⁸Ga-DOTA-TMTP1 was mainly excreted through the renal-urinary route and showed fast clearance from blood.The uptake of ⁶⁸Ga-DOTA-TMTP1 in muscle was relatively low.On microPET images,Hela tumors were clearly visualized within 60 min and the uptake of the radiotracer in Hela tumors was higher than that of C-33A tumors.After blocking with TMTP1,the uptake of Hela tumors was significantly reduced and the specificity of ⁶⁸Ga-DOTA-TMTP1 was thus validated.ConclusionThis study originally employs ⁶⁸Ga labeled TMTP1 peptide for PET imaging in cervical cancer models.⁶⁸Ga-DOTA-TMTP1 was produced rapidly with high yield and high purity and it was found to be a promising PET tracer for targeting highly metastatic cervical cancer exhibiting excellent stability,high specificity and good pharmacokinetic properties.Purpose Since VEGFR-3 plays a critical role in tumor lymphangiogenesis and metastasis,it might become a promising diagnostic indicator and therapeutic target for various solid tumors.TMVP1 is a novel peptide,identified by our research group using bacterial flagella peptide display library,that specifically targets VEGFR-3.TMVP1 can be used for non-invasive imaging and targeted-therapy of VEGFR-3 expressing tumors.Since dimer peptide shows better ability than monomer,we have designed and synthesized a novel radiotracer ⁶⁸Ga-NOTA-?TMVP1?₂ based on TMVP1 homodimer.The purpose of this study was to investigate the biological properties of ⁶⁸Ga-NOTA-?TMVP1?₂ and its clinical application value in tumor-targeted molecular imaging.Methods In this study,we synthesized a TMVP1 homodimer that was conjugated with1,4,7-triazacyclononane-N,N',N'-triacetic acid?NOTA?and labeled with 68 Ga.The quality control of ⁶⁸Ga-NOTA-?TMVP1?₂ was carried out.The in vitro stability study and the octanol-water partition coefficient of ⁶⁸Ga-NOTA-?TMVP1?₂ were determined.We conducted micro PET imaging and biodistribution studies of ⁶⁸Ga-NOTA-?TMVP1?₂ in female athymic nude mice bearing the subcutaneous C-33 A cervical cancer xenografts.After the clinical study was approved by the Institutional Review Board,5 healthy volunteers were recruited to assess the safety and in vivo biodistribution of⁶⁸Ga-NOTA-?TMVP1?₂.⁶⁸Ga-NOTA-?TMVP1?₂ PET/CT imaging and 18F-FDG PET/CT imaging were performed in 22 patients with solid tumors.The uptake of⁶⁸Ga-NOTA-?TMVP1?₂ was analyzed by measuring the SUVmax and SUVmean of lesions and normal muscle tissues.Immunohistochemistry was performed to detect the VEGFR-3 expression level of the lesions and we determined the relationship between⁶⁸Ga-NOTA-?TMVP1?₂ imaging results and receptor expression levels.Results⁶⁸Ga-NOTA-?TMVP1?₂ was successfully synthesized with high radiochemical yield.The radiochemical purity of the product reached over 97% and ⁶⁸Ga-NOTA-?TMVP1?₂showed excellent stability for 3 h.The octanol-water partition coefficient for⁶⁸Ga-NOTA-?TMVP1?₂ was-2.45 ± 0.43 and the tracer is hydrophilic.The in vivo biodistribution experiments demonstrated that ⁶⁸Ga-NOTA-?TMVP1?₂ was excreted mainly through the urinary route and partly through the liver-intestinal route.The tracer was rapidly cleared from the circulation.The specificity of ⁶⁸Ga-NOTA-?TMVP1?₂ was confirmed by the blocking study.⁶⁸Ga-NOTA-?TMVP1?₂ has proven to be safe in all healthy volunteers as well as recruited patients,without any adverse reactions being noticed or reported after intravenous injection of the tracer.The tracer was mainly accumulated in the bladder,kidney and liver,followed by spleen,heart and pancreas and tracers showed the lowest uptake in brain,lung,muscle and red marrow.A total of 22 female patients were enrolled in our study and a majority of the 18F-FDG identified lesions?48/70?showed positive uptake of ⁶⁸Ga-NOTA-?TMVP1?₂.SUVmax and SUVmean were 2.44±1.24 and 1.66±0.94,respectively.With normal muscle?gluteus maximus?as background,tumor-to-background ratio were 3.13±1.81 and 3.62±2.32 based on SUVmax and SUVmean,respectively.Conclusion This study presents a successfully synthesized novel VEGFR-3 receptor imaging agent⁶⁸Ga-NOTA-?TMVP1?₂.The favorable characterizations of ⁶⁸Ga-NOTA-?TMVP1?₂including convenient synthesis,high radioactive yield,high radiochemical purity,excellent stability and specificity warrant its further investigation for clinical cancer imaging.After preclinical research,we conducted clinical transformation trial and initially confirmed its potential in tumor-targeted imaging,which provides feasibility for future clinical applications.