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Introduction: In vivo imaging methods such as Positron Emission Tomography (PET) can be used to examine the relationship between in vitro binding affinity and in vivo occupancy of binding sites in the brain for new drug candidates. In this study, PET imaging in monkey brain was used to evaluate that correlation for a set of four diastereomers of the compound dihydrotetrabenazine (DTBZ), the pharmacologically active metabolite of the drug tetrabenazine., Methods: PET studies of DTBZ diastereomers were completed in a single monkey brain. In vivo occupancies (ED 50 ) were estimated using multiple drug doses and the vesicular monoamine transporter 2 specific radioligand (+)-α-[ 11 C] DTBZ, employing a test-retest sequence of control PET scan, drug administration and a second PET scan completed on a single day., Results: DTBZ has three chiral carbon centers and eight possible stereoisomers, and in vivo occupancy of the target site VMAT2 was observed only for the four diastereomers of DTBZ having the 11bR absolute configuration. The estimated in vivo occupancies (ED 50 values from 0.023 to >3.15 mg/kg) correlated well (R 2  = 0.95) with the in vitro binding affinities (K i values of 4 to 600 nM for the VMAT2), and an even better correlation (R 2  = 0.99) was found for the three isomers with in vitro binding affinities <100 nM., Conclusions: If the physiochemical (MW, log P, pKa) or physiological (metabolism, transport, protein binding) properties of a set of drug stereoisomers are considered similar, the binding affinities determined from in vitro assays may predict the in vivo occupancies of the target binding site in the monkey brain., (Copyright © 2020. Published by Elsevier Inc.)

Objectives: Recently, a deuterated tracer, D6-[ 18 F]FP-(+)-DTBZ, 9-O-hexadeutero-3-[ 18 F]fluoropropoxyl-(+)-dihydrotetrabenazine ([ 18 F]9), targeting vesicular monoamine transporter 2 (VMAT2) in the central nervous system, was reported as a useful imaging agent for the diagnosis of Parkinson's disease (PD). The production of [ 18 F]9 was optimized and simplified by using solid-phase extraction (SPE) purification., Methods: Three major nonradioactive impurities were synthesized and characterized. The preparation of [ 18 F]9 was optimized by using different labeling conditions, and an SPE purification method was evaluated. The influence of chemical impurities in the final dose of [ 18 F]9 was assessed by an in vitro binding assay, an assay of the in vivo biodistribution in mice, and ex vivo and in vitro autoradiography of brain sections., Results: Optimized fluorination conditions for [ 18 F]9 were found - heating at 130 °C for 10 min in DMSO, and a high radiochemical yield and three major chemical impurities were observed. An SPE method involving a Sep-Pak® tC18 Plus Light cartridge with a two-step elution process was successfully implemented. This process gave a good radiochemical yield (38.7 ± 10.5%, decay corrected; radiochemical purity >99%) and low chemical impurities. An in vivo biodistribution study and autoradiography of brain sections showed that there was no significant difference between HPLC-purified and SPE-purified [ 18 F]9., Conclusion: A VMAT2 targeting imaging agent, D6-[ 18 F]FP-(+)-DTBZ, [ 18 F]9, was prepared by optimized labeling conditions and an easy SPE purification. This method offers a short preparation time and operational simplicity. In conjunction with PET imaging, this new VMAT2 agent might be a useful clinical tool for diagnosing PD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Vesicular monoamine transporters 2 (VMAT2) in the brain serve as transporter for packaging monoamine in vesicles for normal CNS neurotransmission. Several VMAT2 imaging agents, [ 11 C]-(+)-DTBZ, dihydrotetrabenazine and [ 18 F]FP-(+)-DTBZ (9-O-fluoropropyl-(+)-dihydro tetrabenazine, a.k.a. [ 18 F]AV-133), are useful for studying the changes in brain function related to monoamine transmission by in vivo imaging. Deuterated analogs have been reported targeting VMAT2 binding sites., Methods: A novel deuterated [ 18 F]9-O-hexaduterofluoropropyl-(+)-dihydrotetrabenazine, [ 18 F]D6-FP-(+)-DTBZ, [ 18 F]1, was prepared as a VMAT2 imaging agent. This 18 F agent which targeted VMAT2 was evaluated by in vitro binding, in vivo biodistribution and microPET imaging studies in rodents., Results: The one step radiolabeling reaction led to the desired [ 18 F]D6-FP-(+)-DTBZ, [ 18 F]1, which showed excellent binding affinity to VMAT2 (Ki=0.32±0.07nM) comparable to that of FP-(+)-DTBZ (Ki=0.33±0.02nM) using [ 18 F]FP-(+)-DTBZ and rat striatum membrane homogenates. In vivo biodistribution in normal rats showed that 1, exhibited excellent brain uptake and comparable high ratio of striatum to cerebellum (target/background) ratio at 1h after injection (ratio of 6.05±0.43 vs 5.66±0.72 for [ 18 F]FP-(+)-DTBZ vs [ 18 F]1, respectively). MicroPET imaging studies in rats further confirm that the striatum with high VMAT2 concentration was clearly delineated in normal rat brain after iv injection of [ 18 F]1. We observed minor changes of metabolism in rat plasma between these two agents; however, the changes showed little effect on regional brain uptake and retention., Conclusions: The results reported here lend support for using [ 18 F]D6-FP-(+)-DTBZ, [ 18 F]1, as in vivo PET imaging agent for VMAT2 binding in the brain., (Copyright © 2017. Published by Elsevier Inc.)

Introduction: The longitudinal reproducibility of in vivo binding potential measures for [ 11 C]dihydrotetrabenazine ([ 11 C]DTBZ) binding to the vesicular monoamine transporter 2 (VMAT2) site in primate brain was examined using a unique dataset of repeated control PET imaging studies., Methods: Forty-one dynamic [ 11 C]DTBZ PET studies were completed in a single rhesus monkey. Imaging equipment (microPET P4), personnel, radiotracer characteristics (injected mass amounts, molar activity) and image data analysis (BP ND-Logan ) were consistent throughout the entire sequence of PET studies., Results: Same day reproducibility of BP ND-Logan estimates of specific binding was very good (-3% and -7% changes) for two control-control sessions. Over the full 74 months, the average BP ND-Logan value for [ 11 C]DTBZ-PET studies was 4.19±0.52, for a variance of 12%. No age-dependent change in binding potentials was observed over the six-year period., Conclusions: If the technical variables associated with PET scanner are consistently maintained, including PET scanner, imaging procedures and radiotracer preparation, in vivo biochemistry can be reproducibly measured in the primate brain over a multi-year period of time., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: 18 F-Fluoropropyl-(+)-dihydrotetrabenazine ( 18 F-FP-(+)-DTBZ) is a vesicular monoamine transporter type 2 (VMAT2) radiotracer for positron emission tomography (PET) imaging to quantify human β-cell mass. Renal cortex and spleen have been suggested as reference regions, however, little is known about 18 F-FP-(+)-DTBZ binding in these regions including the fraction of radiometabolite. We compared the kinetics of 18 F-FP-(+)-DTBZ and its inactive enantiomer 18 F-FP-(-)-DTBZ in baboons, estimated the non-displaceable binding (V ND ) of the tracers, and used ex vivo studies to measure radiometabolite fractions., Methods: PET scans were conducted for up to 4h with (+) and (-) enantiomers. Displacement experiments using unlabeled (+) and (-) enantiomers of FP-DTBZ and fluvoxamine (to evaluate sigma-1 receptor binding) were performed. SUV curves were used to calculate displacement values in the pancreas, renal cortex, and spleen. Distribution volumes (V T ) were computed, and three approaches for calculation of V ND were compared: (1) 18 F-FP-(+)-DTBZ reference V T , (2) 18 F-FP-(-)-DTBZ pancreatic V T , and (3) a scaled 18 F-FP-(+)-DTBZ reference V T values. Ex vivo study was conducted to measure radiometabolite fraction in homogenized tissue samples from baboons at 90min post-injection., Results: Spleen uptake was lowest for both tracers. Highest uptake was in the pancreas with 18 F-FP-(+)-DTBZ and renal cortex with 18 F-FP-(-)-DTBZ. Substantial displacement effect was observed only with unlabeled FP-(+)-DTBZ in the 18 F-FP-(+)-DTBZ studies. Radiometabolite fraction was higher in the renal cortex than the spleen. Approaches (1) and (3) with spleen to estimate V ND provided lowest inter-subject variability of BP ND ., Conclusions: V T differences among organs and between enantiomers indicated that scaling of reference region values is needed for quantification of VMAT2 binding in the pancreas with 18 F-FP-(+)-DTBZ. Since the kidney PET signal has greater partial volume averaging and more radiometabolites, the spleen was considered a more practical candidate for use as a scaled-reference region in the quantification of 18 F-FP-(+)-DTBZ in the pancreas., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: The exact cause(s) of apparent overestimation of β cell mass (BCM) with vesicular monoamine transporter type 2 (VMAT2) PET imaging in type 1 diabetes (T1D) is unknown. The objectives were to estimate in baboons non-displaceable binding of [¹⁸F]fluoropropyl (FP)-(+)-dihydrotetrabenazine (DTBZ) with its inactive enantiomer, [¹⁸F]FP-(-)-DTBZ, to validate the use of the reference tissue (renal cortex or spleen) in VMAT2 quantification; and also to compare specific pancreatic VMAT2 binding with that of the striatum in the same baboon brains because high specific binding signal for the pancreas would be desirable for its accurate quantification., Methods: Baboons (Papio ursinus) had multiple dynamic abdominal and brain PET scans each for 2 h with (+) and (-) enantiomers on separate occasions. Data were analyzed by compartmental models to estimate non-displaceable (V(ND)) and specific (V(S)) VMAT2 binding in respective organs., Results: [¹⁸F]FP-DTBZ PET showed excellent target tissue signal and specific VMAT2 binding in the pancreas (Vs =41±11 mL/cm³) at nearly 80% that of the striatum. Directly estimated non-displaceable binding in the pancreas (V(ND) =12±1 mL/cm³) was similar to that of the renal cortex, spleen or cerebellum., Conclusion: ¹⁸FFP-DTBZ PET shows excellent specific VMAT2 binding in both the pancreas and striatum in baboons. The renal cortex or spleen as the reference tissue in VMAT2 quantification appears supported. However further studies appear warranted to directly estimate pancreatic non-displaceable binding in humans including T1D patients and also to clarify the cause of the apparent overestimation of BCM in T1D., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: Recently, 9-[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ((18)F-AV-133) was reported as a new vesicular monoamine transporter (VMAT2) imaging agent for diagnosis of Parkinson's disease (PD). To shorten the preparation of (18)F-AV-133 and to make it more widely available, we evaluated a simple, rapid purification with a solid-phase extraction method (SPE) using an Oasis HLB cartridge instead of high pressure liquid chromatography (HPLC). The SPE method produced doses containing a pseudo-carrier, 9-hydroxypropyl-(+)-dihydrotetrabenazine (AV-149)., Methods: To test the possible side effects of this pseudo-carrier, comparative dynamic PET scans of the brains of normal monkeys (2 each) and uni-laterally 6-OH-dopamine-lesioned PD monkeys (2 each) were performed using (18)F-AV-133 doses prepared by either SPE (containing pseudo-carrier) or HPLC (containing no pseudo-carrier). Autoradiographs of post mortem monkey brain sections were evaluated to confirm the relative (18)F-AV-133 uptake in the PD monkey brains and the effects of the pseudo-carrier on VMAT2 binding., Results: The radiochemical purity of the (18)F-AV-133, whether prepared by SPE or by HPLC, was excellent (>99%). PET scans of normal and PD monkey brains showed an expected reduction of VMAT2 in the lesioned areas of the striatum. It was not affected by the presence of the pseudo-carrier, AV-149 (maximally 250 μg/dose). The reduced uptake in the striatum of the lesioned monkey brains was confirmed by autoradiography. Ex vivo inhibition studies of (18)F-AV-133 binding in rat brains, conducted with increasing amounts of AV-149, suggested that at the highest concentration (3.5mg/kg) the VMAT2 binding in the striatum was only moderately blocked (20% reduction)., Conclusions: The pseudo-carrier, AV-149, did not affect the (18)F-AV-133/PET imaging of VMAT2 binding sites in normal or uni-laterally lesioned monkey brains. The new streamlined SPE purification method will enable (18)F-AV-133 to be widely available for routine clinical application in determining changes in monoamine neurons for patient with movement disorders or other psychiatric illnesses., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Unlabelled: The vesicular monoamine transporter type II (VMAT2) is highly expressed in pancreatic β-cells and thus has been proposed to be a potential target for measuring β-cell mass (BCM) by molecular imaging. Several tracers based on the TBZ backbone, including 9-fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]AV-133), have shown some promising results as potential biomarkers for BCM despite a relatively high background signal in the pancreas. In the present study, we explore the background binding characteristics of [(18)F]AV-133 in rat pancreas., Methods: Pancreatic exocrine cells and islet cells were isolated and purified from Sprague-Dawley rats. Membrane homogenates, prepared from both pancreatic exocrine and islet cells as well as from brain striatum regions, were used for in vitro binding studies of [(18)F]AV-133 under a selective masking condition. 1,3-Di-o-tolylguanidine (DTG), displaying high and roughly equal affinity for both sigma-1 and sigma-2 receptors, was chosen at 5 μM concentration for the masking/blocking studies., Results: [(18)F]AV-133 binding to rat striatum homogenates was not significantly altered by the presence of DTG. In contrast, [(18)F]AV-133 showed significant competition with DTG for binding sites in rat pancreatic exocrine homogenates as well as in rat islet cell homogenates. Importantly, in the presence of DTG, [(18)F]AV-133 showed a single high-affinity binding site on islet cell homogenates with a K(d) value of 3.8 nM which is consistent with the affinity reported previously for VMAT2 sites in rat pancreas., Conclusions: [(18)F]AV-133, in addition to a high-affinity VMAT2 binding site, binds with low affinity (but high capacity) to sigma components that are present in the rat pancreas. Identification of the cause of background binding of [(18)F]AV-133 to rat pancreatic tissue may lead to improved methods for quantification., (Copyright © 2011 Elsevier Inc. All rights reserved.)

(+)-α-[(11)C]Dihydrotetrabenazine ((+)-[(11)C]DTBZ), a radioligand for the vesicular monoamine transporter type 2 (VMAT2), has been previously proposed as an in vivo marker of beta-cell degeneration in the pancreas. The stereospecificity of uptake of [(11)C]DTBZ into rat pancreas was examined here using radiolabeled forms of the (+)- and (-)-isomers. Pancreas localization of (+)-[(11)C]DTBZ could be partially blocked by prior administration of unlabeled (+)-DTBZ. Pancreatic uptake of the (-)-isomer was unexpectedly high and could not be blocked by pretreatment with (+)-DTBZ, but could be significantly reduced by treatment with racemic tetrabenazine, an in vivo source of (-)-DTBZ. These studies indicate that the inactive isomer of DTBZ does not provide a mechanism for defining the nonspecific binding of (+)-DTBZ in rat pancreas., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Introduction: The sensitivity of the in vivo binding of [(11)C]dihydrotetrabenazine ([(11)C]DTBZ) and [(11)C]methylphenidate ([(11)C]MPH) to their respective targets - vesicular monoamine transporter type 2 (VMAT2) and neuronal membrane dopamine transporter - after alterations in endogenous levels of dopamine was examined in the rat brain., Methods: In vivo binding of [(11)C]DTBZ and [(11)C]MPH was determined using a bolus+infusion protocol. The in vitro number of VMAT2 binding sites was determined by autoradiography., Results: Repeated dosing with alpha-methyl-p-tyrosine (AMPT) at doses that significantly (-75%) depleted brain tissue dopamine levels resulted in increased (+36%) in vivo [(11)C]DTBZ binding to VMAT2 in the striatum. The increase in binding could be completely reversed via treatment with L-DOPA/benserazide to restore dopamine levels. There were no changes in the total number of VMAT2 binding sites, as measured using in vitro autoradiography. No changes were observed for in vivo [(11)C]MPH binding to the dopamine transporter in the striatum following AMPT pretreatment., Conclusion: These results indicate that large reductions in dopamine concentrations in the rat brain can produce modest but significant changes in the binding of radioligands to VMAT2, which can be reversed by replenishment of dopamine using exogenous L-DOPA., (Copyright 2010. Published by Elsevier Inc.)

Labeling derivatives of dihydrotetrabenazine (DTBZ) with F-18 (T(1/2)=110 min) instead of C-11 (T(1/2)=20 min) would improve their utility and availability for imaging vesicular monoamine transporters (VMAT2) in clinical settings. The successful synthesis, reported previously, of two novel 9-fluoroalkyl(+/-)-DTBZ ligands prompted us to study the optically resolved active ligand 9-fluoropropyl-(+)-DTBZ (FP-(+)-DTBZ), which may have more promising characteristics. The inhibition constant (K(i)) estimated for FP-(+)-DTBZ (using [(3)H](+/-)-DTBZ as the labeled ligand in rat striatal homogenates) showed a lower value as compared to the racemic FP-(+/-)-DTBZ (0.10+/-0.01 vs 0.19+/-0.04 nM). The inactive isomer, FP-(-)-DTBZ, displayed a much lower binding affinity with a K(i) value >3000 nM. Biodistribution studies in mice after an iv injection of [(18)F]FP-(+)-DTBZ exhibited a ratio of striatum (ST, target) to cerebellum (CB, background) of 4.51 at 30 min postinjection, which is a higher value than previously obtained with the racemic ligand [(18)F]FP-(+/-)-DTBZ (ST/CB=2.95). Brain extraction at 30 min after the tracer injection in mice showed that >95% of the radioactivity corresponded to the parent, nonmetabolized, compound remaining in the ST, suggesting that the tracer has an excellent in vivo stability. Furthermore, localization of the tracer in the brain examined with ex vivo autoradiography displayed a typical distribution pattern consistent with VMAT2 sites. The highest labeling was observed in monoaminergic neuron regions (caudate putamen, olfactory tubercle, nucleus accumbens, substantia nigra, dorsal raphe and locus coerules). We also tested the selective labeling of this tracer at the dopamine neurons in unilateral-lesioned mice (treated with 6-hydroxydopamine). When [(18)F]FP-(+)-DTBZ and [(125)I]IPT ((N-(3'-iodopropen-2'-yl)-2-beta-carbomethoxy-3-beta-(4-chlorophenyl)tropane, a selective marker for dopamine transporters (DATs) in dopaminergic neurons) were simultaneously injected into lesioned mice, we observed an excellent correlation (r=0.95) for these tracers. From these findings, we conclude that [(18)F]FP-(+)-DTBZ is a sensitive and selective tracer for VMAT2 binding sites and it may be useful for in vivo evaluation of diseases relating to changes of monoamine neuronal integrity.

[11C]Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9-[11C]methoxy-10 -methoxy-1,2,3,4,6,7,- hexahydro-11bH-bezo[alpha]-quinolizine) ([11C]DTBZ) was synthesized by reacting the 9-hydroxy precursor in DMSO with gas-phase [11C]methyl iodide on a column of alumina impregnated with KOH. The reaction was instantaneous at room temperature. This column was then connected to the inlet of a short column containing basic alumina. Elution with cyclohexane removed radioactive contaminants. The radioactive product was then eluted with a few milliliters ether containing 1% ethanol. The [11C]DTBZ was obtained in isolated yields of > 200 mCi and specific activities > 1600 Ci/mmol.