Synthesis and characterization of [I]TZ6544, a promising radioligand for investigating sphingosine-1-phosphate receptor 2.

Introduction: Sphingosine-1-phosphate receptor 2 (S1PR2) activation exerts a critical role in biological abnormalities and diseases. A suitable radiotracer will advance our understanding of S1PR2 pathophysiology of diseases. The objective of this study is to evaluate the potential of iodine-125 labeled [I]TZ6544 to be used for screening new compounds binding toward S1PR2, and assessing the changes of S1PR2 expression in the kidney of streptozotocin-induced diabetic rats.

Methods: [I]TZ6544 was synthesized from borate precursor by copper (II)-catalyzed iodization reaction with [I]NaI. [I]TZ6544 was characterized using human recombinant S1PR2 cell membrane and biodistribution studies of []TZ6544 were performed on Wistar rats that were euthanized at 5 and 30 min post-injection. A rat model of diabetes was induced by IV injection of streptozotocin (55 mg/kg). In vitro autoradiography studies, immunostaining, and enzyme-linked immunosorbent assay (ELISA) analysis were performed in both diabetic and control rats.

Results: Radiosynthesis of [I]TZ6544 was achieved successfully with good radiochemical yields of ~47% and high radiochemical purity of >99%. [I]TZ6544 is a potent ligand in vitro for S1PR2 with K value of 4.31 nM. [I]TZ6544 and [P]-labeled endogenous S1P provided comparable IC values in radioactive competitive binding assays against known S1PR2 ligands. Compared to control, the kidney of diabetic rats had increased uptake of [I]TZ6544, which could be reduced by a S1PR2 antagonist, JTE-013. Immunostaining and ELISA analysis confirmed that the diabetic rat had increased S1PR2 expression in the kidney.

Conclusions: [I]TZ6544 was synthesized successfully in high yields, and in vitro evaluation suggested [I]TZ6544 has high potential to be used for screening new S1PR2 compounds and investigating the pathophysiology of S1PR2 functions. The availability of [I]TZ6544 may facilitate the development of therapeutics and imaging agents targeting S1PR2. ADVANCES IN KNOWLEDGE: [I]TZ6544 showed increased expression of S1PR2 in diabetic rat kidney and can be used to determine binding potency of S1PR2 compounds.