Synthesis And Evaluation Of Novel PET Molecular Probes Based On Polyamine Metabolism

Objective:Polyamines are involved in many fundamental processes of cell growth and transformation,and intracellular polyamine levels are tightly regulated.Studies have shown that polyamines and polyamine synthase are significantly elevated in tumor cells compared to normal cells.The diagnosis and treatment targeting key enzymes and transporters of the polyamine metabolic pathway has become an important strategy for cancer management.Ornithine decarboxylase（ODC）is the first rate-limiting enzyme of the polyamine synthesis pathway,and its expression and activity directly affect the polyamine biosynthesis.ODC is closely related to the development of tumors and is considered an important biomarker for the diagnosis and treatment of tumors targeting polyamine metabolism.In the first part of this study,we designed and synthesized a⁶⁸Ga-labeled ornithine([⁶⁸Ga]Ga-NOTA-Orn),which was labeled using Ga-68 after linking the ornithine to the macrocyclic chelator NOTA.The presence of upregulated polyamine transporter system（PTS）expression in most tumor cells makes PTS an important target for tumor diagnosis and treatment.In the second part of this study,a novel molecular probe[⁶⁸Ga]Ga-NOTA-Spermine targeting PTS was prepared.We systematically evaluated the potential of[⁶⁸Ga]Ga-NOTA-Orn for non-invasive visualization detecting expression levels of ODC in tumor tissues and as a tumor diagnostic imaging agent through cellular uptake and competitive inhibition experiments,biodistribution studies,and in vivo PET imaging of tumor models,and the feasibility of[⁶⁸Ga]Ga-NOTA-Spermine as a novel molecular probe for tumor diagnosis.Methods:An ornithine derivative NOTA-Bn-NCS-Orn was prepared as a precursor by solid-phase synthesis using the bifunctional chelator NOTA-Bn-NCS and ornithine as raw materials.The molecular probe[⁶⁸Ga]Ga-NOTA-Orn was prepared by labeling the precursor with the Ga-68.The molecular probe[⁶⁸Ga]Ga-NOTA-Spermine was prepared by modifying natural spermine using the macrocyclic chelator NOTA to obtain a spermine analogue as a precursor,which was then labeled with the Ga-68.The stability of the probes in serum and saline was determined using Radio-HPLC.We use a variety of tumor cell lines to study the uptake properties and transport mechanisms of molecular probes by cells.The distribution of the probes in normal mice was studied.AR42J,A549and DU145 tumor-bearing mice were cultured for PET/CT in vivo imaging.Evaluation the expression of ODC in tumor tissues and major organs were performed by immunohistochemical staining.Results:The total synthesis time of[⁶⁸Ga]Ga-NOTA-Orn is about 30 min,the labeling rate is45-50%（uncorrected）,the radiochemical purity is>98%,and the stability in vivo and in vitro is great.Cellular uptake and competitive inhibition assays suggest that the probe transports in a similar manner to L-ornithine and may further interact with the ODC upon entry into the tumor cell.Biodistribution and PET imaging studies have shown that[⁶⁸Ga]Ga-NOTA-Orn is mainly metabolized by the kidney,with low distribution in all other tissues and organs throughout the body,and can be specifically uptake by ODC-positive tumors with a high target to non-target（T/NT=8.8）ratio.The[⁶⁸Ga]Ga-NOTA-Spermine has a preparation time of about 30 min,a labeling rate of 50-55%（uncorrected）,a radiochemical purity of>98%,and is very stable in vivo and in vitro.The results of biodistribution and PET imaging showed that[⁶⁸Ga]Ga-NOTA-Spermine is mainly metabolized by the kidney,with a low distribution in other tissues and organs throughout the body,and can be specifically taken up by tumors with a high target to non-target（T/NT=3.88）ratio.Conclusion:In this study,two molecular probes based on polyamine metabolism,[⁶⁸Ga]Ga-NOTA-Orn and[⁶⁸Ga]Ga-NOTA-Spermine,were successfully prepared and systematically biologically evaluated.The experimental results showed that both probes have great potential to become novel molecular probes for nuclear medicine tumor diagnosis,and[⁶⁸Ga]Ga-NOTA-Orn has the potential for non-invasive visualization of ODC expression levels in tumor tissues.