A Unique ¹⁸F-labeled G-protein-coupled receptor 44 (GPR44) radiotracer: design, radio-synthesis and evaluation in the rodents

Objectives: The G-protein-coupled receptor 44 (GPR44) plays a key role in the inflammatory diseases, and emerged as a therapeutic target in asthma and other allergic disorders. In human pancreas, GPR44 is expressed in the islets of Langerhans and not in the exocrine pancreas. GPR44 seems a reliable biomarker for assessment of islets mass, which is significant in diabetes and related diseases. However, F-18 tracer targeting GPR44 has not been reported. Of relevance, most of the reported ligand for GPR44 displays a free carboxylic acid group. Even the ¹⁸F labeling method was well developed, labeling compound including free carboxyl group in on step is still challenging. Given the short half-live of ¹⁸F, labeling compound including a free carboxyl group in one step is highly desirable. The purpose of this study was to develop an ¹⁸F GPR44 tracer, and to evaluate the tracer ex vivo in rodents.

Methods: TM30089 is a selective GPR44 antagonist with excellent potency (GPR44 Ki = 0.6 nM). Radionuclide incorporation using ¹⁸F-fluoride was accomplished through nucleophilic substitution of a novel precursor derived from TM30089. The labeling experiments were preformed using the GE TRACERlab™ FN radio-synthesis module. Precursor was prepared from commercially available starting material and [¹⁸F] labeling optimized through modification in temperatures, reaction solvents and reaction times. The final product was purified by reverse phase semi-preparative HPLC. The expression of GPR44 in human islets was validated by immunofluorescence staining and Western blot. Bio-distribution studies were conducted in adult male NOD/SCID mice to determine tracer distribution. Animals were euthanized at 30, 60 and 90 minutes post-injection of [¹⁸F] Ab-1. Organs of interest were collected, weighed, and counted on an automated gamma counter. Uptake of radioactivity was calculated as percentage injected dose per gram (%I.D./g).

Results: Quaternary ammonium salt precursor 8 was identified and obtained in 7 steps from commercially available starting materials. Precursor 8 was converted to [¹⁸F] Ab-1 in 15 minutes through a one-step labeling reaction. The product [¹⁸F] Ab-1 was purified by HPLC in 15.5 minutes with RCY's of ~30% and high radiochemical purity (> 98%). Immunofluorescence staining and Western blot analysis confirmed that GPR44 is expressed in isolated human islets. Initial bio-distribution studies and blocking experiment showed that low tracer uptake in murine pancreases, consistent with low expression of GPR44 in mice pancreas from other reports.

Conclusions: The expression of GPR44 in human pancreases islets was confirmed. A novel tracer GPR44-targeting radiolabeled tracer, [¹⁸F] Ab-1, was produced with a synthetic protocol that gave good yield and radiochemical purity. Initial bio-distribution studies showed minimal tracer uptake in murine pancreases. Studies are ongoing to evaluate the potential of the tracer to track human islets transplanted into rodents. Further studies of [¹⁸F] Ab-1 will assess its application to beta cell mass imaging, as a biomarker for human beta cell-derived cancers.