In vivo validation of novel GLUT-1 targeting click-conjugates

Objectives The Warburg effect describes proliferating cancer cells that have higher metabolic requirements than normal cells. High uptake of glucose by cancer cells is compensated by overexpression of glucose transporters, especially GLUT1 and hexokinase enzyme. Here, we present our results on In vivo validation of our lead candidates; glucosamine-click conjugates (RMX-11-Gc and RMX-08-Gc) as new PET imaging agents that target the metabolically active cancer cells.

Methods RMX-Gc conjugates were synthesized by coupling of 2-acetamido-N-(ϵ-minocaproyl)-2-deoxy-β-D-glucosylamine to MFCO-NHS click or BNCO-NHS, respectively followed by Cu-free conjugation with 3-azido-propyl-monoamide DOTA. Both compounds were analyzed by ESI-MS and HPLC. Their ⁶⁸Ga-labeling proceeded in 0.5M NaOAc pH=4.1 at 95^oC for 10min (⁶⁸Ge/⁶⁸Ga generator (ITG GmbH Germany) and purified using C18 Sep-pak cartridges. The percent radiochemical yields (RCYs) of labeling reactions were determined by iTLC and radio-HPLC. ⁶⁸Ga-labeled agent were validated as PET metabolic agents in xenograft model of breast cancer (SKBR-3) using Genisys 4 microPET in a static imaging mode at 0min, 30min, 60min and 90min post injection.

Results Both ⁶⁸Ga-labeled RMX-Gc conjugates have shown excellent tumor-specific accumulation with no uptake by normal organs (liver, brain, lung or spleen) and relatively fast elimination through kidneys. RMX-GC-08 showed significantly higher activity in tumor than RMX-GC-11 with maximum activity accumulated in tumor at 90min Vs. at 60min for RMX-GC-11 as quantified by ROI analysis of PET images.

Conclusions Both ⁶⁸Ga-labeled RMX-GC (RMX-08-Gc and RMX-11-Gc) agents have shown high potential for imaging of GLUT-1 overexpressing tumors. Minor discrepancy in their pharmacokinetic properties can be potentially explained by differences in their lipophilic properties and MW. (* S.T., D.R and I.T. contributed equally).