⁶⁸Ga-labeled click conjugate targeting GLUT1-transporters

Objectives Copper-free click conjugation has been successfully applied for the synthesis of imaging and therapeutic agents with high yield and purity. Our goal was to evaluate this approach for the synthesis of glucose-based imaging agents.

Methods Synthesis of cyclooctyne-glucosamine conjugate 1 was performed by coupling of MFCO-NHS to 2-acetamido-N-(ϵ-aminocaproyl)-2-deoxy-β-D-glucosylamine. The conjugate 1 reacted with 3-azido-propyl-monoamide DOTA using Cu-free click reaction. Products 1 and 2 were analyzed by ESI-MS and HPLC. ⁶⁸Ga-labeling was performed in 0.5M NaOAc pH=4.1 at 95^oC for 10min (⁶⁸Ge/⁶⁸Ga generator ITG) and analyzed using ITLC and radio-HPLC. Radiochemical stability was determined by their incubation in PBS (1X), DMEM media, and FBS. ⁶⁸Ga-labeled agent was study in vitro in 13762 cancer cell line using ⁶⁸Ga-DOTA as a standard. Competition uptake studies were performed in the presence of GLUT-1 targeting molecules, glucose and cytochalasin B.

Results The ESI spectrum of compound 1 and 2 showed expected [m/z+Na] peaks of 621.6 (predicted 598.7) and [m/z+H]=1095.2 (predicted 1094), respectively. ⁶⁸Ga-radiolabeling of compound 1 proceed with RCY > 99% and in vitro stability greater than 97.2± 1.1% intact in PBS and 95.5± 0.9% intact in media at 2 h by iTLC. Cellular uptake of ⁶⁸Ga-compound 2 was 2.7± 0.1 %ID/mg in 13762 and 0.2 %ID/mg of ⁶⁸Ga-DOTA. This uptake was blocked over 67% in the presence of cold glucose and cytoclasin B.

Conclusions GLUT1 targeting click conjugates were successfully synthesized and labeled with high radiochemical yield and stability and evaluated as potential GLUT1-targeting imaging agents