Purpose The aim of this study was to optimize the radiolabeling method of [ 18 F]fluoropropyl-(+)-dihydrotetrabenazine ([ 18 F]FP-(+)-DTBZ) to fulfill the demand of preclinical and clinical application. Procedures Optimized labeling conditions were performed by altering the molar ratio of precursor to base (P/B), base species, solvents, reaction temperature, reaction time, and precursor concentration through manual radiosynthesis of [ 18 F]FP-(+)-DTBZ. The conditions with the highest radiochemical yield (RCY) were applied to automated radiosynthesis, and the crude product was purified with a Sep-Pak Plus C18 cartridge. Quality control and stability of [ 18 F]FP-(+)-DTBZ were carried out by HPLC. In vitro cellular uptake and blocking assays were conducted in human neuroblastoma cell line SH-SY5Y. In vivo imaging with small animal positron emission tomography (microPET) was performed with Sprague–Dawley rats. Results Under the optimized conditions (P/K 2 CO 3 = 1:8, heating at 120 °C for 3 min in dimethyl sulfoxide), an RCY of 88.7 % was obtained with 1.0 mg precursor. The optimized reaction conditions were successfully applied to an automated module and gave a high activity yield (AY) of 30–55 % in about 40 min with a > 99.0 % radiochemical purity (RCP) and a > 44.4 GBq/μmol molar activity ( A m ). Stability test displayed that the RCP retained > 98.0 % in 8 h in saline and in phosphate buffer saline (PBS, pH 7.4). In vitro cellular uptake assay showed accumulation of [ 18 F]FP-(+)-DTBZ in SH-SY5Y cells, which could be significantly inhibited by vesicular monoamine transporter 2 (VMAT2) inhibitor DTBZ. MicroPET images of rat brain displayed that the striatum showed the highest uptake with a standardized uptake value (SUV) of 3.91 ± 0.30 at ~ 70 min. Co-injection with DTBZ (1.0 mg/kg) resulted in a 75 % decrease of the striatal SUV, confirming the specificity of [ 18 F]FP-(+)-DTBZ to VMAT2. Conclusions We obtained an optimized radiolabeling method of [ 18 F]FP-(+)-DTBZ and successfully applied it to a commercial available module. The automated synthesis gave a high AY and RCP of [ 18 F]FP-(+)-DTBZ with high and specific binding to VMAT2, facilitating its routine application for VMAT2 tracing.
[ 18 F]fluoropropyl-(+)-dihydrotetrabenazine ([ 18 F]FP-(+)-DTBZ) is a rising positron tracer for imaging vesicular monoamine transporter II (VMAT2) in the central nervous system. The present work was to develop a novel chromatographic method capable of the molar activity (A m ) determination of [ 18 F]FP-(+)-DTBZ. As a complement work of the A m measurement, we also investigated the effect of A m on the quantitative analysis of VMAT2 autoradiography with [ 18 F]FP-(+)-DTBZ. The A m determination was performed by high performance liquid chromatography (HPLC) using the non-radioactive standard (FP-(+)-DTBZ) for calibration plot of peak area against concentration. Based on this correlation, the A m of [ 18 F]FP-(+)-DTBZ was calculated and corrected to the end of synthesis. In the quantitative analysis of in vitro VMAT2 autoradiography, the striatum radioactivity uptake together with the uptake ratio of striatum versus cortex reduced along with the decrease of A m and the increase of the FP-(+)-DTBZ content. Therefore, the A m and the corresponding FP-(+)-DTBZ content have a significant effect on the quantitative analysis of VMAT2 autoradiography using [ 18 F]FP-(+)-DTBZ.
Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington’s Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.
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