A Novel Potential Positron Emission Tomography Imaging Agent for Vesicular Monoamine Transporter Type 2

In the early 1990s, 9-(+)-11C-dihydrotetrabenazine (9-(+)-11C-DTBZ) was shown to be a useful positron emission tomography (PET) imaging agent for various neurodegenerative disorders. Here, we described the radiosynthesis and evaluation of the 9-(+)-11C-DTBZ analog, 10-(+)-11C-DTBZ, as a vesicular monoamine transporter 2 (VMAT2) imaging agent and compare it with 9-(+)-11C-DTBZ. 10-(+)-11C-DTBZ was obtained by 11C-MeI methylation with its 10 hydroxy precursor in the presence of 5 M NaOH. It had a slightly better average radiochemical yield of 35.3 ± 3.6% (decay-corrected to end of synthesis (EOS)) than did 9-(+)-11C-DTBZ (30.5 ± 2.3%). MicroPET studies showed that 10-(+)-11C-DTBZ had a striatum-to-cerebellum ratio of 3.74 ± 0.21 at 40 min post-injection, while the ratio of 9-(+)-11C-DTBZ was 2.50 ± 0.33. This indicated that 10-(+)-11C-DTBZ has a higher specific uptake in VMAT2-rich brain regions, and 10-(+)-11C-DTBZ may be a potential VMAT2 radioligand. Our experiment is the first study of 10-(+)-11C-DTBZ to include dynamic brain distribution in rat brains.


Introduction
Vesicular monoamine transporter 2 (VMAT2), a member of the solute carrier family 18 with 12 transmembrane domains, is the protein responsible for transporting monoamine neurotransmitters (dopamine, norepinephrine, serotonin) into synaptic vesicles for subsequent storage and release [1,2]. VMAT2 abnormalities have been implicated in a variety of neurodegenerative disorders, including Parkinson's and Huntington's diseases [3,4]. VMAT2 also has been found to be highly expressed in human pancreas beta cells, which are related to diabetes [5][6][7][8], as well as in the central nervous system. However, the relationship between VMAT2 and the diseases mentioned previously or their underlying causes remains unclear.
Positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging of VMAT2 would further our understanding of its pathophysiology. PET is a non-invasive and highly sensitive technique that enables imaging of a live body using appropriate radiotracers and facilities. The resulting images could reflect the distribution and density of the target, which could provide valuable information regarding both the target and its relationship with diseases in the body. Given the strengths of PET, a specific PET tracer would be helpful in evaluating the body as well as the brain on a molecular level.
Currently, the radionuclides frequently used in PET are fluorine-18 and carbon-11, which have half-lives of 109 and 20 min, respectively. Although carbon-11 has a much shorter half-life, which limits its feasibility, it is still a useful radionuclide in clinical research because it allows multiple imaging sessions within one day. Therefore, studies of two or more protein targets in the same biological pathway are feasible after a short delay when using 11 C-labeled radiotracers.

Materials and Methods General
(TBZ) to obtain (+)-9-O-desmethyl-DTBZ or (+)-10-O-desmethyl-DTBZ. TBZ derivatives (Fig 1) were synthesized in the laboratory of the School of Pharmacy (National Taiwan University, Taipei, Taiwan). Sodium hydroxide was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trifluoroacetic acid was purchased from Alfa Aesar (Ward Hill, MA, USA). Analytical reagent-grade reagents and solvents were purchased from Aldrich or Merck. The tC 18 Sep-Pak and Sep-Pak Light QMA cartridges were acquired from Waters Chromatography Division, Millipore Corporation.
HPLC analysis was performed with a Waters HPLC system equipped with both UV (280 nm) and radioactivity detectors.

Animal preparation
Rat studies were performed using 250-330 g male Sprague Dawley rats (BioLASCO Taiwan Co., Ltd.). All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee. This work was approved by the Laboratory Animal Center of National Taiwan University College of Medicine. The animals were housed and handled according to institutional guidelines. All animals were starved overnight prior to the experiment. On the day of the study, rats were anesthetized using 5.0% isoflurane. Each rat was positioned on the scanner bed, and anesthesia was applied using a nose cone. A transmission scan was acquired. A radiotracer (0.8-1.3 mCi) was injected intravenously into the tail vein of the rat. Isoflurane was reduced and maintained at 2.0% following injection.

Radiochemistry
All steps (Fig 2) up to sterile filtration of the final product solution were performed using the integrated functions of the GE TRACERlab FXc synthesis system. The reaction of gaseous 11 C-methyl iodide with (2R, 3R, 11bR)-10-O-desmethyldihydrotetrabenazine or (2R, 3R, 11bR)-9-O-desmethyldihydrotetrabenazine took place in the FXc reaction vessel. The vessel was charged with 2.0 mg of precursor, 15 μL of 5 M NaOH and 400 μL of dimethylformamide. 11 C-Methyl iodide was extracted from the methyl iodide synthesis unit and introduced into the reaction vessel at a fixed flow rate of 15 mL/min for 3 min. The flow was then stopped, and the solution was stirred for an additional 2 min. The reaction mixture was diluted with 0.6 mL of ethanol/50 mM of NH 4 OAc adjusted to pH = 4.5 by acetic acid (10:90); injected into a preparatory HPLC column (Waters XTerra Prep, 10 μm, 10 x 150 mm); and eluted with 10/90 ethanol/ 50 mM of NH 4 OAc adjusted to pH = 4.5 by acetic acid at 6 mL/min. The product fraction was collected at 9.5 min, diluted with isotonic saline and passed through a 0.22-μm sterilizing filter into a sterile 10-mL multi-dose vial.

Quality control
Chemical and radiochemical impurities were detected using HPLC. For a quality assessment with analytical HPLC analysis, a reversed-phase C 18 column (Phenomenex Gemini, 5 μm, 4.6 x 250 mm) with acetonitrile:50 mM of NH 4 OAc adjusted to pH = 4.5 by acetic acid (20:80) as the eluent was used with a flow rate of 1 mL/min. The retention times of 9-(+)-11 C-DTBZ and 10-(+)-11 C-DTBZ were 9.8 and 10.5 min, respectively, and both radiotracers were further confirmed with authentic standards for radiochemical identities.

MicroPET imaging
For the animal study, fasting male Sprague Dawley rats (n = 3) were used. The rats had free access to water for 12 hours prior to the experiment and were then injected with a bolus of 0.8-1.3 mCi of radiotracer. A small-animal Argus PET/CT scanner was used to produce dynamic sinograms for 90 min with 4 x 15 sec, 2 x 30 sec, 4 x 60 sec, 3 x 180 sec, 5 x 300 sec, 3 x 600 sec and 1 x 1200 sec frames.
Image analyses of the rat brain were performed with PMOD, version 3.7 (Sciffer, PMOD Technologies Ltd.). First, rat brain images were aligned to a standard template of a rat brain using PMOD's rigid matching tool. The region of interests of each rat brain were then manually defined and expressed as standard uptake values (SUVs). The specific uptake ratios (SURs) of the striata were expressed as striatum/ cerebellum.

Results and Discussion
PET tracers designed to target specific neurochemical processes offer new possibilities for improving the differentiation of various central nervous system-related disorders, such as dementia [2]. Therefore, because of the physiological importance of VMAT2, we proposed to synthesize 10-(+)-11 C-DTBZ, the regioisomer of 9-(+)-11 C-DTBZ, which has been widely used for VMAT2 imaging, and we performed a comparative in vivo evaluation of both radiotracers.

Author Contributions
Conceived and designed the experiments: LWH.