NIR760-XLP6, a novel near-infrared fluorescence imaging probe that preferentially binds to cannabinoid receptors CB₂R over CB₁R.

Objectives The type 2 cannabinoid receptors (CB₂R) have important regulatory role in a host of pathophysiological processes. However, its exact biological function remains unclear and could be better studied with highly sensitive and selective imaging tools for identifying the receptors. The problem is not only the challenge in distinguishing CB₂R and CB₁R with molecular ligands due to the high sequence homology between the two, but also on how to couple a fluorescent dye to a CB₂R ligand without compromising the binding affinity. In this study, we prepared near-infrared fluorescence imaging probe NIR760-XLP6 that binds preferentially to CB₂R over CB₁R.

Methods First, the selectivity and specificity of NIR760-XLP6 were demonstrated using DBT-CB₂ and DBT-CB₁ cells in vitro. Then, the probe was injected in four groups of mice via tail vein: (1) mice with DBT-CB₂ tumor were treated with NIR760-XLP6, (2) mice with DBT-CB₁ tumor were treated with NIR760-XLP6, (3) mice with DBT-CB₂ tumor were first treated with XLP4 as blocking and then treated with NIR760-XLP6, (4) mice with DBT-CB₂ tumor were treated with free dye NIR760. Finally, ex vivo study were utilized to confirm the finding.

Results In fluorescence microscopy studies, remarkably higher fluorescence from the NIR760-XLP6 was observed in DBT-CB₂ cells than in DBT-CB₁ cells. In mouse tumor models, 48 and 72 h post injection, stronger fluorescence signals in the tumor area were observed in NIR760-XLP6 treated DBT-CB₂ group than that in the DBT-CB₁group. Lastly, in ex vivo biodistribution study, 2-fold higher tumor contrast was observed in the DBT-CB₂ tumor mice treated with NIR760-XLP6 than that in the DBT-CB₁ tumor mice.

Conclusions We have successfully developed a novel NIR fluorescent probe, NIR760-XLP6, which preferentially binds to CB₂R over CB₁R both in vitro and in vivo.

Research Support This work was supported by a startup fund provided by the Department of Radiology, University of Pittsburgh. This project used the UPCI imaging facilities supported, in part, by award P30CA047904.