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Vesicular monoamine transporter 2 (VMAT2) is crucial for packaging monoamine neurotransmitters into synaptic vesicles, with their dysregulation linked to schizophrenia, mood disorders, and Parkinson's disease. Tetrabenazine (TBZ) and valbenazine (VBZ), both FDA-approved VMAT2 inhibitors, are employed to treat chorea and tardive dyskinesia (TD). Our study presents the structures of VMAT2 bound to substrates serotonin (5-HT) and dopamine (DA), as well as the inhibitors TBZ and VBZ. Utilizing cryo-electron microscopy (cryo-EM), mutagenesis functional assays, and molecular dynamics (MD) simulations, we elucidate the mechanisms of substrate transport and drug inhibition. Our MD simulations indicate potential binding poses of substrate (5-HT) in both cytosol-facing and lumen-facing states, emphasizing the significance of protonation of key acidic residues for substrate release. We demonstrate that TBZ locks VMAT2 in a lumen-facing occluded state, while VBZ stabilizes it in a lumen-facing conformation. These insights enhance our understanding of VMAT2 function and provide valuable insights for the development of novel therapeutic strategies for psychiatric disorders., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Human vesicular monoamine transporter 2 (VMAT2), a member of the SLC18 family, plays a crucial role in regulating neurotransmitters in the brain by facilitating their uptake and storage within vesicles, preparing them for exocytotic release. Because of its central role in neurotransmitter signalling and neuroprotection, VMAT2 is a target for neurodegenerative diseases and movement disorders, with its inhibitor being used as therapeutics. Despite the importance of VMAT2 in pharmacophysiology, the molecular basis of VMAT2-mediated neurotransmitter transport and its inhibition remains unclear. Here we show the cryo-electron microscopy structure of VMAT2 in the substrate-free state, in complex with the neurotransmitter dopamine, and in complex with the inhibitor tetrabenazine. In addition to these structural determinations, monoamine uptake assays, mutational studies, and pKa value predictions were performed to characterize the dynamic changes in VMAT2 structure. These results provide a structural basis for understanding VMAT2-mediated vesicular transport of neurotransmitters and a platform for modulation of current inhibitor design., (© 2024. The Author(s).)

Vesicular monoamine transporter 2 (VMAT2) accumulates monoamines in presynaptic vesicles for storage and exocytotic release, and has a vital role in monoaminergic neurotransmission 1-3 . Dysfunction of monoaminergic systems causes many neurological and psychiatric disorders, including Parkinson's disease, hyperkinetic movement disorders and depression 4-6 . Suppressing VMAT2 with reserpine and tetrabenazine alleviates symptoms of hypertension and Huntington's disease 7,8 , respectively. Here we describe cryo-electron microscopy structures of human VMAT2 complexed with serotonin and three clinical drugs at 3.5-2.8 Å, demonstrating the structural basis for transport and inhibition. Reserpine and ketanserin occupy the substrate-binding pocket and lock VMAT2 in cytoplasm-facing and lumen-facing states, respectively, whereas tetrabenazine binds in a VMAT2-specific pocket and traps VMAT2 in an occluded state. The structures in three distinct states also reveal the structural basis of the VMAT2 transport cycle. Our study establishes a structural foundation for the mechanistic understanding of substrate recognition, transport, drug inhibition and pharmacology of VMAT2 while shedding light on the rational design of potential therapeutic agents., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood 1 . Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders 2 . Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission 3-5 . VMATs are also therapeutic drug targets for a number of different conditions 6-9 . Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Aim: To separate and evaluate 9-cyclopropylmethoxy-dihydrotetrabenazine (13a) and its stereoisomers for their high affinity for vesicular monoamine transporter-2 (VMAT2). Method: Stereoisomers of 13a were separated and configurations were ascertained by chiral chromatography and crystal diffraction combined with 1 H- 1 H NOESY assay. Possible binding modes of eight stereoisomers and VMAT2 were explored by molecular docking assays. The VMAT2 affinity of the stereoisomers, inhibition in vivo  and pharmacokinetics in rats were evaluated. Results: Three stereoisomers were obtained: P1, P2 and P3, and all had similar VMAT2 binding modes. P2 [(2 R , 3 R , 11b R )-13a] showed the highest potential VMAT2 binding activity ( K i  = 0.75 nM), decreased locomotor activity in rats and had an oral absolute bioavailability of 92.0%. Conclusion: P2 has good efficacy and pharmacokinetic properties and warrants further development to treat tardive dyskinesia.

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.)

There are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2ga as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers.

Purpose: Diabetic retinopathy (DR) is characterized by dopaminergic neuron loss in the retina of the eyes. [ 18 F]fluoropropyl-(+)-dihydrotetrabenazine ([ 18 F]FP-(+)-DTBZ) positron emission tomography (PET) has been shown to detect dopaminergic neuron loss. The study is to investigate the feasibility of PET imaging with [ 18 F]FP-(+)-DTBZ for early diagnosis of diabetic retinopathy (DR) in diabetes mellitus (DM) rat models., Methods: The DM rat model was established by a single intraperitoneal injection of streptozotocin (STZ) (65 mg/kg). After 4 weeks, 8 weeks, and 12 weeks of STZ injection, the retinas of the rats were evaluated by electroretinogram (ERG), color fundus photography (CFP), fundus fluorescein angiography (FFA), and small animal PET scan with [ 18 F]FP-(+)-DTBZ by targeting vesicular monoamine transporter 2 (VMAT2). [ 18 F]FP-(+)-DTBZ uptake in retina was quantified as standardized uptake value (SUV). Immunofluorescence staining and Western blot were also performed to confirm the expression level of VMAT2 in retina., Results: ERG dysfunction was observed at 8 weeks in STZ-diabetic rats, evidenced by smaller amplitudes of oscillatory potentials (OPs) when compared with OPs in normal rats. CFP and FFA showed no significant difference in vascular leakage and neovascularization between STZ-diabetic retinas and normal ones until 8 weeks. PET imaging revealed that the SUV of [ 18 F]FP-(+)-DTBZ was significantly lower in the STZ-diabetic retinas compared with the normal ones as early as of week 4. The results from immunofluorescence staining and Western blots confirmed the early findings in PET imaging studies., Conclusions: Early DR can be non-invasively detected with PET imaging using [ 18 F]FP-(+)-DTBZ targeting VMAT2. The expression level of VMAT2 in retina may act as a new biomarker for early DR diagnosis.

The hydrogen isotopes deuterium (D) and tritium (T) have become essential tools in chemistry, biology and medicine 1 . Beyond their widespread use in spectroscopy, mass spectrometry and mechanistic and pharmacokinetic studies, there has been considerable interest in incorporating deuterium into drug molecules 1 . Deutetrabenazine, a deuterated drug that is promising for the treatment of Huntington's disease 2 , was recently approved by the United States' Food and Drug Administration. The deuterium kinetic isotope effect, which compares the rate of a chemical reaction for a compound with that for its deuterated counterpart, can be substantial 1,3,4 . The strategic replacement of hydrogen with deuterium can affect both the rate of metabolism and the distribution of metabolites for a compound 5 , improving the efficacy and safety of a drug. The pharmacokinetics of a deuterated compound depends on the location(s) of deuterium. Although methods are available for deuterium incorporation at both early and late stages of the synthesis of a drug 6,7 , these processes are often unselective and the stereoisotopic purity can be difficult to measure 7,8 . Here we describe the preparation of stereoselectively deuterated building blocks for pharmaceutical research. As a proof of concept, we demonstrate a four-step conversion of benzene to cyclohexene with varying degrees of deuterium incorporation, via binding to a tungsten complex. Using different combinations of deuterated and proteated acid and hydride reagents, the deuterated positions on the cyclohexene ring can be controlled precisely. In total, 52 unique stereoisotopomers of cyclohexene are available, in the form of ten different isotopologues. This concept can be extended to prepare discrete stereoisotopomers of functionalized cyclohexenes. Such systematic methods for the preparation of pharmacologically active compounds as discrete stereoisotopomers could improve the pharmacological and toxicological properties of drugs and provide mechanistic information related to their distribution and metabolism in the body.

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.

Tetrabenazine is a US Food and Drug Administration (FDA)-approved drug that exhibits a dopamine depleting effect and is used for the treatment of chorea in Huntington's disease. Mechanistically, tetrabenazine binds and inhibits vesicular monoamine transporter type 2, which is responsible for importing neurotransmitters from the cytosol to the vesicles in neuronal cells. This transportation contributes to the release of neurotransmitters inside the cell to the synaptic cleft, resulting in dopaminergic signal transmission. The highly potent inhibitory activity of tetrabenazine has led to its advanced applications and in-depth investigation of prodrug design and metabolite drug discovery. In addition, the synthesis of enantiomerically pure tetrabenazine has been pursued. After a series of research studies, tetrabenazine derivatives such as valbenazine and deutetrabenazine have been approved by the US FDA. In addition, radioisotopically labeled tetrabenazine permits the early diagnosis of Parkinson's disease, which is difficult to treat during the later stages of this disease. These applications were made possible by the synthetic efforts aimed toward the efficient and asymmetric synthesis of tetrabenazine. In this review, various syntheses of tetrabenazine and its derivatives have been summarized., Competing Interests: The authors declare no conflict of interest.

Ethnopharmacological Relevance: In Turkish folk medicine, infusions and decoctions prepared from the flowers, fruits and aerial parts of Centaurea kurdica Reichardt (Asteraceae) are used as sedative and antidepressant-like effects of various sedative plants have been identified in many studies. The present study was designed to evaluate the antidepressant activity of this plant., Materials and Methods: n-Hexane, ethyl acetate (EtOAc), and methanol (MeOH) extracts were prepared from the branches with leaves and also flowers of the plant. Antidepressant potentials of these extracts were researched by using the forced swimming test, tail suspension test, and antagonism of tetrabenazine-induced ptosis, hypothermia, and suppression of locomotor activity., Results: After determination of high antidepressant potentials of MeOH extract prepared from flowers and n-hexane extract prepared from branches with leaves, isolation studies were carried out on these two extracts and the main active components were determined as β-amyrin, mixture of β-sitosterol and stigmasterol and costunolide for the branches with leaves and quercitrin, isoquercetin and naringenin-7-O-glucopyranoside for the flowers., Conclusions: As a result of the mechanistic and toxicity studies planned on this plant, it is thought that C. kurdica may be a glimmer of hope for depressed patients., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.)

Objective: Stable heavy isotopes of hydrogen, carbon, and other elements have been incorporated into drug molecules, largely as tracers for quantitation during the drug development process. Studies involving the human use of drugs labeled with deuterium suggest that these compounds may offer some advantages when compared with their nondeuterated counterparts. Deuteration has gained attention because of its potential to affect the pharmacokinetic and metabolic profiles of drugs. Deutetrabenazine (Austedo, Teva Pharmaceutical Industries, Ltd) is the first deuterated drug to receive Food and Drug Administration approval. This deuterated form of the drug tetrabenazine is indicated for the treatment of chorea associated with Huntington's disease as well as tardive dyskinesia. Ongoing clinical trials suggest that a number of other deuterated compounds are being evaluated for the treatment of human diseases and not merely as research tools., Data Sources: A search of the MEDLINE (1946 to present) database was undertaken using the Ovid interface. The search was conducted using the heading deuterium and then limited to Administration & Dosage, Adverse Effects, Pharmacokinetics, Pharmacology, Poisoning, Therapeutic Use, and Toxicity., Study Selection and Data Extraction: All articles were reviewed and those with human information were included. Review articles were likewise interrogated for additional published human data., Conclusions: Deuterated compounds may, in some cases, offer advantages over nondeuterated forms, often through alterations in clearance. Deuteration may also redirect metabolic pathways in directions that reduce toxicities. The approval of additional deuterated compounds may soon follow. Clinicians will need to be familiar with the dosing, efficacy, potential side effects, and unique metabolic profiles of these new entities.

The basal ganglia and dopaminergic pathways play a central role in hyperkinetic movement disorders. Vesicular monoamine transporter 2 (VMAT2) inhibitors, which deplete dopamine at presynaptic striatal nerve terminals, are a class of drugs that have long been used to treat hyperkinetic movement disorders, but have recently gained more attention following their development for specific indications in the United States. At present, there are three commercially available VMAT2 inhibitors: tetrabenazine, deutetrabenazine, and valbenazine. Pharmacokinetics, metabolism, and dosing vary significantly between the three drugs, and likely underlie the more favorable side effect profile of the newer agents (deutetrabenazine and valbenazine). Tetrabenazine and deutetrabenazine have demonstrated safety and efficacy in the treatment of chorea associated with Huntington's disease, including in randomized controlled trials, although direct comparison studies are limited. Both deutetrabenazine and valbenazine have demonstrated safety and efficacy in the treatment of tardive dyskinesia, with multiple double-blind, placebo-controlled trials, whereas tetrabenazine has been studied less rigorously. There have been no blinded, prospective trials with tetrabenazine in Tourette's syndrome (TS); however, double-blind, placebo-controlled trials in TS are ongoing for both deutetrabenazine and valbenazine. Given the favored side effect profile of newer VMAT2 inhibitors, clinicians should be aware of the distinctions between agents and become familiar with differences in their use, especially as there is potential for their utilization to increase across the range of hyperkinetic movement disorders.

Purpose: Previous studies demonstrated the utility of [ 18 F]fluoropropyl-(+)-dihydrotetrabenazine ([ 18 F]FP-(+)-DTBZ) as a positron emission tomography (PET) radiotracer for the vesicular monoamine transporter type 2 (VMAT2) to quantify beta cell mass in healthy control (HC) and type 1 diabetes mellitus (T1DM) groups. Quantification of specific binding requires measurement of non-displaceable uptake. Our goal was to identify a reference tissue (renal cortex or spleen) to quantify pancreatic non-specific binding of [ 18 F]FP-(+)-DTBZ with the inactive enantiomer, [ 18 F]FP-(-)-DTBZ. This was the first human study of [ 18 F]FP-(-)-DTBZ., Procedures: Six HCs and four T1DM patients were scanned on separate days after injection of [ 18 F]FP-(+)-DTBZ or [ 18 F]FP-(-)-DTBZ. Distribution volumes (V T ) and standardized uptake values (SUVs) were compared between groups. Three methods for calculation of non-displaceable uptake (V ND ) or reference SUV were applied: (1) use of [ 18 F]FP-(+)-DTBZ reference V T as V ND , assuming V ND is uniform across organs; (2) use of [ 18 F]FP-(-)-DTBZ pancreatic V T as V ND , assuming that V ND is uniform between enantiomers in the pancreas; and (3) use of a scaled [ 18 F]FP-(+)-DTBZ reference V T as V ND , assuming that a ratio of non-displaceable uptake between organs is uniform between enantiomers. Group differences in V T (or SUV), binding potential (BP ND ), or SUV ratio (SUVR) were estimated using these three methods., Results: [ 18 F]FP-(-)-DTBZ V T values were different among organs, and V T (+) and V T (-) were also different in the renal cortex and spleen. Method 3 with the spleen to estimate V ND (or reference SUV) gave the highest non-displaceable uptake and the largest HC vs. T1DM group differences. Significant group differences were also observed in V T (or SUV) with method 1 using spleen. SUV was affected by differences in the input function between groups and between enantiomers., Conclusions: Non-displaceable uptake was different among organs and between enantiomers. Use of scaled spleen V T values for V ND is a suitable method for quantification of VMAT2 in the pancreas.

Purpose: To investigate the relationship between expression level of vesicular monoamine transporter 2 (VMAT2) and myopia, as well as the feasibility of noninvasive myopia diagnosis through imaging VMAT2 in retina by using [ 18 F]fluoropropyl-(+)-dihydrotetrabenazine ([ 18 F]FP-(+)-DTBZ)., Procedures: The right eyes of ten guinea pigs were deprived of vision to establish form-deprived (FD) myopia and the left eyes were untreated as the self-control eyes. The location and expression level of VMAT2 in the eyes were detected by micro-positron emission tomography (PET)/X-ray computed tomography (CT) imaging through using [ 18 F]FP-(+)-DTBZ. Immunofluorescence staining and Western blot were used to confirm the location and expression level of VMAT2 in the eyes. The concentrations of dopamine (DA) and its metabolites including 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were also investigated by high-performance liquid chromatography., Results: The right eyes deprived of vision were obviously myopic (- 3.17 ± 1.33 D) after procedure, while the left eyes were hyperopic (4.60 ± 0.83 D, P < 0.0001). The main expressions of VMAT2 in the eyes were located in retina. VMAT2 was significantly reduced in the myopic retina compared to the normal one from PET/CT results (P = 0.0008), which could also be verified by Western blots (P = 0.029). The concentrations of DA, DOPAC, and HVA in the FD eyes were all significantly less than those in the control eyes (P = 0.024, P = 0.018, P = 0.008). As a role of storing and releasing DA in vesicles, VMAT2 was demonstrated positively correlating with the amounts of DA (P = 0.030), DOPAC (P = 0.038), and HVA (P = 0.025) through Pearson's correlation coefficient test., Conclusions: We demonstrate that [ 18 F]FP-(+)-DTBZ can be used to noninvasively image VMAT2 in retina. The expression level of VMAT2 in retina may act as a new biomarker for myopia diagnosis. The decreasing of VMAT2 expression level may play an important role in the development of myopia through correspondingly reducing the amount of DA in retina.

Cassette dosing is also known as N-in-One dosing: several compounds are simultaneously administrated to a single animal and then the samples are rapidly detected by LC-MS/MS. This approach is a successful strategy to enhance the efficiency of drug discovery and reduce animal usage. However, no report on the utility of the cassette approach in radiotracer discovery has appeared in the literature. This study designed a cassette microdose with LC-MS/MS method to enhance the throughput for screening radiopharmaceutical biodistribution in the rat brain directly. Three unradiolabeled compounds (FPBM FPBM2 and AV-133) were chosen as model drugs administrated intravenously to the rats as a cassette as opposed to discrete study. The rat brain biodistribution data, target localization, the differential uptake ratio (%ID/g) and the brain tissue-specific binding ratio were obtained by the LC-MS/MS analysis. These data matched very well with the values obtained by the standard radioactivity measurements. Moreover, no significant differences between discrete dosing and cassette dosing were observed. By circumventing the need for radiolabeled molecules, this method may be high-throughput and safe for the research and development of new PET imaging agents. The combination of cassette microdosing and LC-MS/MS would be a medium throughput screening tool at an early stage in the discovery/development process of PET imaging agents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tardive dyskinesia (TD) encompasses the spectrum of iatrogenic hyperkinetic movement disorders following exposure to dopamine receptor-blocking agents (DRBAs). Despite the advent of atypical or second- and third-generation antipsychotics with a presumably lower risk of complications, TD remains a persistent and challenging problem. Prevention is the first step in mitigating the risk of TD, but early recognition, gradual withdrawal of offending medications, and appropriate treatment are also critical. As TD is often a persistent and troublesome disorder, specific antidyskinetic therapies are often needed for symptomatic relief. The vesicular monoamine transporter 2 (VMAT2) inhibitors, which include tetrabenazine, deutetrabenazine, and valbenazine, are considered the treatment of choice for most patients with TD. Deutetrabenazine-a deuterated version of tetrabenazine-and valbenazine, the purified parent product of one of the main tetrabenazine metabolites, are novel VMAT2 inhibitors and the only drugs to receive approval from the US FDA for the treatment of TD. VMAT2 inhibitors deplete presynaptic dopamine and reduce involuntary movements in many hyperkinetic movement disorders, particularly TD, Huntington disease, and Tourette syndrome. The active metabolites of the VMAT2 inhibitors have high affinity for VMAT2 and minimal off-target binding. Compared with tetrabenazine, deutetrabenazine and valbenazine have pharmacokinetic advantages that translate into less frequent dosing and better tolerability. However, no head-to-head studies have compared the various VMAT2 inhibitors. One of the major advantages of VMAT2 inhibitors over DRBAs, which are still being used by some clinicians in the treatment of some hyperkinetic disorders, including TD, is that they are not associated with the development of TD. We also briefly discuss other treatment options for TD, including amantadine, clonazepam, Gingko biloba, zolpidem, botulinum toxin, and deep brain stimulation. Treatment of TD and other drug-induced movement disorders must be individualized and based on the severity, phenomenology, potential side effects, and other factors discussed in this review.

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.)

The dopaminergic system plays a key role in the central nervous system, regulating executive function, arousal, reward, and motor control. Dysregulation of this critical monoaminergic system has been associated with diseases of the central nervous system including schizophrenia, Parkinson's disease, and disorders such as attention deficit hyperactivity disorders and addiction. Drugs that modify the dopaminergic system by modulating the activity of dopamine have been successful in demonstrating clinical efficacy by providing treatments for these diseases. Specifically, antipsychotics, both typical and atypical, while acting on a number of monoaminergic systems in the brain, primarily target the dopamine system via inhibition of postsynaptic dopamine receptors. The vesicular monoamine transporter 2 (VMAT2) is an integral presynaptic protein that regulates the packaging and subsequent release of dopamine and other monoamines from neuronal vesicles into the synapse. Despite acting on opposing sides of the synapse, both antipsychotics and VMAT2 inhibitors act to decrease the activity of central dopaminergic systems. Tardive dyskinesia is a disorder characterized by involuntary repetitive movements and thought to be a result of a hyperdopaminergic state precipitated by the use of antipsychotics. Valbenazine (NBI-98854), a novel compound that selectively inhibits VMAT2 through an active metabolite, has been developed for the treatment of tardive dyskinesia and is the first drug approved for the treatment of this disorder. This chapter describes the process leading to the discovery of valbenazine, its pharmacological characteristics, along with preclinical and clinical evidence of its efficacy., (© 2018 Elsevier B.V. All rights reserved.)

Background: The use of single-photon emission computed tomography and positron emission tomography (PET) has proven to be helpful in differentiating Parkinson's disease (PD) from other movement disorders with a sensitivity of up to 95%., Objective: The objective of this study was to determine the accuracy of [11C]DTBZ PET imaging in patients with clinically uncertain parkinsonism from a tertiary referral center in Mexico City., Materials and Methods: Patients who underwent [11C]DTBZ PET brain scan due to clinically uncertain parkinsonism where divided into two groups: PD or non-PD. A scan was considered positive when visual assessment revealed a decrease in [11C]DTBZ uptake typical for PD; a scan was considered negative when visual assessment showed no decrease in [11C]DTBZ uptake or showed a decrease in tracer uptake in a non-PD pattern. Sensitivity, specificity, and positive and negative predictive values were calculated using a 2 × 2 table, with a 95% confidence interval., Results: A total of 39 patients were included in the study. 14 PET studies were deemed positive and 25 PET studies were deemed negative; 12 true positives and 23 true negatives were found. This yielded a sensitivity of 92.9% (95% CI, 66.1-99.8), specificity of 92% (95% CI, 74-99), PPV of 86.7% (95% CI, 63.1-96.1), and NPV of 95.8% (95% CI, 79.1-98.4)., Conclusions: The [11C]DTBZ PET has an excellent accuracy for differentiating idiopathic PD from other disorders., (Copyright: © 2017 SecretarÍa de Salud.)

Background: The feasibility of beta cell mass (BCM) imaging and quantification with positron emission tomography (PET) in the pancreas is controversial. In an effort to shed some light on this topic, we have used a xenograft model of rat insulinoma (RIN) in mice, mimicking an intramuscular islet transplantation situation., Methods: A total of 105 RIN cells were subcutaneously implanted in nude mice (N.=8). Tumor size and glycaemia levels were determined daily. Rat C-peptide was measured to demonstrate rat insulin production. PET imaging with 11C-(+)-α-dihydrotetrabenazine (11C-DTBZ) was done at 3 and 4 weeks and compared with 18F-FDG and 18F-DOPA studies in the same mice. Ex-vivo autoradiography with 11C-DTBZ was carried out in frozen sections of tumors. VMAT2 expression was measured by Western-blot and immunohistochemistry in tumors and RIN cells., Results: Functional rat insulin production in mice was demonstrated by substantial decrease in glycaemia (<50 mg/dL by week 4) and rat C-peptide levels (7.2±2.6 ng/mL) similar to those measured in control rats. PET studies showed that tumor imaging with 11C-DTBZ at four (N.=8) and five (N.=5) weeks was negative; only bigger tumors could be seen with 18F-DOPA. In explanted tumors 11C-DTBZ autoradiography was negative, albeit VMAT2 expression measured by Western-blot and immunohistochemistry was lower than in cultured RIN cells., Conclusions: Although insulinomas are fully functional it does not seem feasible to use 11C-DTBZ for in-vivo measuring of BCM. This might either be due to inherent technical limitations of PET, decrease in VMAT2 expression in the tumors due to unknown reasons, or other biological limiting facts.

Introduction: The pharmacokinetics and/or toxicity of many known drugs can be modified by selective deuteration, an area of significant commercial interest and scientific and regulatory progress. Areas covered: This review firstly discusses recent developments in deuterated drugs including the FDA approval of deutetrabenazine. Secondly, it discusses 35 U.S.C. §103 'obviousness' as it relates to recent patent prosecution, and also to Inter Partes Review (IPR). IPR is a new post-award review of patentability under §102 or §103, two IPR petitions upon deuterated drugs have been instituted and included §103 arguments. Finally, an extended analysis of §103 obviousness based upon the practices of major pharmaceutical companies is provided, that supports rather late priority dates, while §102 is also discussed. Expert opinion: The total value of transactions involving deuterated drugs is close to $5 billion. While the importance of §103 'obviousness' rejections remains in patent applications under current prosecution, IPR of issued patents is developing and will affect likely affect §103 interpretations in this area. However, patents are still issuing with later priority dates, and further litigation will likely occur.

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.)

Background: Tetrabenazine (TBZ) activity is thought to result from four isomeric dihydrotetrabenazine (HTBZ) metabolites ([+]-α-HTBZ, [-]-α-HTBZ, [+]-β-HTBZ, [-]-β-HTBZ). Each isomer has a unique profile of vesicular monoamine transporter 2 (VMAT2) inhibition and off-target binding. Previously published data only report total isomer (α) and (β) concentrations. We developed a method to quantify the individual HTBZ isomers in samples from patients with Huntington's disease receiving TBZ. For comparison, concentrations of [+]-α-HTBZ, the single active metabolite shared by valbenazine (VBZ) and TBZ, were determined in samples from patients with tardive dyskinesia receiving VBZ., Methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the four individual HTBZ isomers. Concentrations were determined in serum from patients with Huntington's disease administered TBZ and plasma from patients with tardive dyskinesia administered VBZ once daily., Results: In patients administered TBZ, [-]-α-HTBZ and [+]-β-HTBZ were the most abundant HTBZ isomers while [-]-β-HTBZ and [+]-α-HTBZ were present as minor metabolites. Only [+]-α-HTBZ was observed in patients administered VBZ., Conclusions: Based on relative abundance and potency, [+]-β-HTBZ appears to be the primary contributor to VMAT2 inhibition by TBZ, a finding in contrast with the generally held assertion that [+]-α-HTBZ is the major contributor. [-]-α-HTBZ, the other abundant TBZ metabolite, has much lower VMAT2 inhibitory potency than [+]-β-HTBZ, but increased affinity for other CNS targets, which may contribute to off-target effects of TBZ. In contrast, pharmacological activity for VBZ is derived primarily from [+]-α-HTBZ. Individual HTBZ isomer concentrations provide a more clinically relevant endpoint for assessing on- and off-target effects of TBZ than total isomer concentrations.

Valbenazine (Ingrezza™) is an orally bioavailable, selective, vesicular monoamine transporter 2 (VMAT2) inhibitor being developed by Neurocrine Biosciences for the treatment of various central nervous system disorders. Valbenazine has been approved in the USA for the treatment of adults with tardive dyskinesia (TD), is at various stages of development in other countries for TD and is in phase 2 development in the USA for Tourette syndrome. This article summarizes the milestones in the development of valbenazine leading to its first global approval in the USA for the treatment of adults with TD.

Objectives: Orodispersible films (ODF) were formulated to facilitate tetrabenazine (TBZ) administration to paediatric population for the treatment of hyperkinetic movement disorders., Methods: ODF were obtained by solvent casting/evaporation method using four different polymers (HPMC, PVP, pullulan and HEC). Physicochemical, mechanical and biopharmaceutical characterizations as well as API state in ODF by thermal analysis were investigated to define and compare formulations. ODF stability was also monitored during 6 months to follow evolution of properties., Key Findings: Analyses at T0 showed few differences between formulations: results of physicochemical and mechanical characterizations were almost similar for each formulation and TBZ appeared at the amorphous state in all cases. ODF delivery system allowed a major improvement of TBZ dissolution profile in buccal conditions compared with pure drug. However, after 3 and 6 months of stability, a TBZ recrystallization occurred for formulations based on PVP and HEC associated with a decrease of drug release in saliva conditions., Conclusions: HPMC-ODF (F1) appeared as the best formulation. Indeed, physicochemical, mechanical and biopharmaceutical characteristic remained intact. In addition, TBZ remained in amorphous state during stability study., (© 2016 Royal Pharmaceutical Society.)

In this work, the formation and stability of amorphous solid dispersions (SDs) as orodispersible films (ODF) were investigated using tetrabenazine (TBZ) as a poorly water soluble drug. The influence of polymer nature and pH-modifier incorporation to form and maintain SDs was investigated. TBZ-loaded ODF were formulated using 4 different polymers (HPMC, PVP, Pullulan, and HEC). Binary systems (BS) were obtained mixing the drug with different polymers, while ternary (TS) systems were also obtained by adding citric acid to solubilize TBZ in the mixture. Drug dissolution studies, thermal analysis and X-ray diffraction were carried out to characterize the physical state of API in ODF. ODF made of TS allowed a major improvement of TBZ dissolution profile in buccal conditions compared to a pure drug or BS. DSC and X-ray diffraction revealed that API was in amorphous state in TS while remained crystalline in BS. Following 6 months of storage, TBZ recrystallization occurred for PVP-TS and HEC-TS which induced a decrease of drug release in saliva conditions. HPMC and PUL-TS maintained API in amorphous state during 6 months. Briefly, amorphous SDs were obtained by the pre-dissolution of the drug in acidified water and incorporation in polymeric films. The miscibility and potential interaction between TBZ and polymers have been identified as important factor to explain stability differences., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Deutetrabenazine is a derivative of tetrabenazine in which two trideuteromethoxy groups substitute two methoxy groups. The active metabolites of deutetrabenazine have a longer half-life than those of tetrabenazine, together with a greater overall absorption. However, the peak plasma concentrations are lower. Because of these pharmacokinetic differences, deutetrabenazine can be given twice daily, thus improving compliance. The lower peak concentrations may account for a lower incidence of some unwanted adverse effects. Unlike tetrabenazine, deutetrabenazine has no effect on the QT interval. Treatment with deutetrabenazine significantly improved chorea in Huntington's disease, the hyperkinetic features of tardive dyskinesia, and tics in Tourette syndrome. In all three conditions, deutetrabenazine produced an acceptable level of overall adverse effects without causing any severe adverse effects., (Copyright 2017 Clarivate Analytics.)

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.)

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., Competing Interests: The authors have declared that no competing interests exist.

Our research groups recently described a series of small-molecule inducers of β-cell proliferation that could be used to increase β-cell mass. To mitigate the risk of nonspecific proliferation of other cell types, we devised a delivery strategy built on the tissue specificity observed in the experimental β-cell imaging agent (+)-dihydrotetrabenazine (DTBZ). The β-cell proliferator agent aminopyrazine (AP) was covalently linked with (+)-DTBZ to afford conjugates that retain both the proliferation activity and binding affinity for vesicular monoamine transporter-2 (VMAT2). In vivo mouse tissue distribution studies of a prototypical AP-DTBZ conjugate showed 15-fold pancreas exposure over plasma. Tissue-to-plasma ratios in liver and kidneys were two- and five-fold, respectively. This work is the first demonstration of enhanced delivery of β-cell-proliferating molecules to the pancreas by leveraging the intrinsic tissue specificity of a β-cell imaging agent., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

(±)-Tetrabenazine was synthesized in six steps from commercially available compounds. The key cyclization substrate was assembled rapidly via Baylis-Hillman and aza-Michael reactions. Annulation of the final ring was achieved through visible light photocatalysis, wherein carbon-carbon bond formation was driven by the oxidation of a tertiary amine. Solvent played a critical role in the photoredox cyclization outcome, whereas methanol led to a mixed ketal, acetonitrile/water (10:1) gave direct cyclization to (±)-tetrabenazine and occurred more rapidly.

Many drugs are carbon-based, and carbon-hydrogen bonding is particularly relevant for understanding important properties of drug molecules. Deuteration refers to the selective replacement of protium hydrogen isotope atoms in small-molecule drugs with deuterium hydrogen isotope atoms. Deuteration of a drug is most likely to affect pharmacokinetic properties, such as metabolism, rather than its pharmacodynamic effects. For this reason, the metabolism of certain drugs may be favorably influenced when deuterium is substituted for protium, resulting in improved safety, tolerability, or efficacy. Examples of deuterated drugs that have been evaluated in clinical studies include paroxetine, tetrabenazine, and dextromethorphan., (Copyright 2015, SLACK Incorporated.)

Dihydrotetrabenazine (DTBZ) derivatized from (+) Tetrabenazine (TBZ) has been used for imaging the expression of VMAT2 when labeled with (11)C (t1/2=20.3 min) or (18)F (t1/2=110 min) in neurodegenerative diseases or pancreatic beta-cell. Because (11)C or (18)F radiolabels are only available in the proximity of a biomedical cyclotron facility, here we report our work of derivatizing (+) and (-) DTBZ using a (64)Cu-specific bifunctional chelator scaffold ((64)Cu: t1/2=12.7 h) for the preparation of long-lived VMAT2 targeted radiotracers, (64)Cu-CB-TE2A-(+)-DTBZ and (64)Cu-CB-TE2A-(-)-DTBZ. The specific VMAT2 binding affinity of (64)Cu-CB-TE2A-(+)-DTBZ measured using rat brain homogenate or porcine islets was not compromised by our chemical modifications while that of its (-) counterpart remained low as in (11)C or (18)F labeled (±) DTBZ., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

During solution formulation study of tetrabenazine (TBZ), a dopamine depleting agent, used in chorea associated with Huntington's disease and symptomatic treatment of hyperkinetic movement disorder it was observed a strong discoloration upon storage. We investigated this physico-chemical behavior by implementing forced degradation studies. It was observed yellowing only under Suntest(®) light exposure of TBZ solution. LC-MS (liquid chromatography coupled to mass spectrometer detection) analysis of light exposed TBZ samples allowed us to propose 1,11b-dedihydrotetrabenazine (DTBZ) and 1,3,4,11b-detetrahydrotetrabenazine (TTBZ) as the main TBZ impurities. Synthesis and complete structural determination of DTBZ and TTBZ·HCl by NMR and X-ray crystallography were carried out. They were identical in LC-MS with polar impurities found in light exposed TBZ samples. However, even if these TBZ degradation products are correlated with discoloration of TBZ solution there is no evidence they are directly responsible of it., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic β cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying β-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated β-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into β cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived β cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of β-cell differentiation and its application to a cost-effective production of functional β cells for cell therapy.

Purpose: Respiratory motion during PET∕CT imaging can cause substantial image blurring and underestimation of tracer concentration for both static and dynamic studies. In this study, the authors developed an event-by-event respiratory motion correction method that used three-dimensional internal-one-dimensional external motion correlation (INTEX3D) in listmode reconstruction. The authors aim to fully correct for organ/tumor-specific rigid motion caused by respiration using all detected events to eliminate both intraframe and interframe motion, and investigate the quantitative improvement in static and dynamic imaging., Methods: The positional translation of an internal organ or tumor during respiration was first determined from the reconstructions of multiple phase-gated images. A level set (active contour) method was used to segment the targeted internal organs/tumors whose centroids were determined. The mean displacement of the external respiratory signal acquired by the Anzai system that corresponded to each phase-gated frame was determined. Three linear correlations between the 1D Anzai mean displacements and the 3D centroids of the internal organ/tumor were established. The 3D internal motion signal with high temporal resolution was then generated by applying each of the three correlation functions to the entire Anzai trace (40 Hz) to guide event-by-event motion correction in listmode reconstruction. The reference location was determined as the location where CT images were acquired to facilitate phase-matched attenuation correction and anatomical-based postfiltering. The proposed method was evaluated with a NEMA phantom driven by a QUASAR respiratory motion platform, and human studies with two tracers: pancreatic beta cell tracer [(18)F]FP(+)DTBZ and tumor hypoxia tracer [(18)F]fluoromisonidazole (FMISO). An anatomical-based postreconstruction filter was applied to the motion-corrected images to reduce noise while preserving quantitative accuracy and organ boundaries in the patient studies., Results: The INTEX3D method yielded an increase of 5%-9% and 32%-40% in contrast recovery coefficient on the hot spheres in the NEMA phantom, compared to the reconstructions with only 1D motion correction (INTEX1D) and no motion correction, respectively. The proposed method also increased the mean activities of the pancreas and kidney by 9.3% and 11.2%, respectively, across three subjects in the FPDTBZ studies, and the average lesion-to-blood ratio by 20% across three lesions in the FMISO study, compared to the reconstructions without motion correction. In addition, the proposed method reduced intragate motion as compared to phase-gated images. The application of the anatomical-based postreconstruction filter further reduced noise in the background by >50% compared to reconstructions without postfiltering, while preserving quantitative accuracy and organ boundaries. Finally, the measurements of the time-activity curves from a subject with FPDTBZ showed that INTEX3D yielded 18% and 11% maximum increases in tracer concentration in the pancreas and kidney cortex, respectively., Conclusions: These results suggest that the proposed method can effectively compensate for both intragate and intergate respiratory motion while preserving all the counts, and is applicable to dynamic studies.

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of tetrabenazine and its active metabolites α-dihydrotetrabenazine and β-dihydrotetrabenazine in human plasma. Tetrabenazine d7 was used as internal standard (IS). The analytes were extracted from 200 μL aliquots of human plasma via solid-phase extraction using C18 solid-phase extraction cartridges. The reconstituted samples were chromatographed on a Zorbax SB C18 column using a 60:40 (v/v) mixture of acetonitrile and 5 mm ammonium acetate as the mobile phase at a flow rate of 0.8 mL/min. The API-4000 LC-MS/MS in multiple reaction-monitoring mode was used for detection. The calibration curves obtained were linear (r(2) ≥ 0.99) over the concentration range of 0.01-5.03 ng/mL for tetrabenazine and 0.50-100 ng/mL for α-dihydrotetrabenazine and β-dihydrotetrabenazine. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies., (Copyright © 2013 John Wiley & Sons, Ltd.)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a-i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [(3)H]-DTBZ binding (Ki=560 nM), with 15-fold greater affinity for this site than GZ-793A (Ki=8.29 μM). Analog 11f also showed similar potency of inhibition of [(3)H]-DA uptake into vesicles (Ki=45 nM) compared to that for GZ-793A (Ki=29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function., (Copyright © 2013. Published by Elsevier Ltd.)

This article reports, for the first time, on the absolute configuration of (+)-9-benzyloxy-α-dihydrotetrabenazine (8), as determined from the perspective of X-ray crystallography. Compound 8 was prepared by a six-step reaction using 3-benzyloxy-4-methoxybenzaldehyde (1) as a starting material. The X-ray crystal diffraction structure of two compounds, racemic 9-benzyloxy-tetrabenazine (5) and the diastereomeric salt of compound 8, is also described for the first time in this article. The X-ray results and the chiral HPLC helped elucidate that compound 8 has an absolute configuration as 2R,3R,11bR. The crystal structure of racemic compound 5 contains two symmetry- independent molecules in the unit cell. Interestingly, while they are structural isomers, they are enantiomers, too, i.e., in solution, because they are not mirror images of each other in the crystal lattice. In order to elucidate the intermolecular interaction mechanism of the diastereomeric salt of compound 8, its crystal packing was investigated with regard to the weak interactions, such as salt bridge, OH...O and CH...O hydrogen bonds, and intermolecular CH...π interaction. The results showed that the carbonyl-assisted salt bridges and the OH...O hydrogen bonds formed polar columns in the crystal structure of the diastereomeric salt of compound 8, resembling butterflies with open wings as viewed along the c-axis. These polar columns were extended to three-dimensional network by intermolecular CH...O hydrogen bonds and intermolecular CH...π interactions., (Copyright © 2013 Wiley Periodicals, Inc.)

A mixed micellar liquid chromatographic (LC) method, the mobile phase consisting of anionic and non-ionic surfactants, has been developed for the high-speed direct radiometabolite analysis of positron emission tomography (PET) radioligands in plasma. The addition of Triton X-100 on an anionic surfactant sodium dodecyl sulphate (SDS) mobile phase improved elution strength and peak efficiency for many PET radioligands. Several radioligands could be easily separated from their radioactive metabolites with short run time of only 4 min using a "pure" (without organic solvent) mixed micellar mobile phase and semi-preparative monolithic C(18)-bonded silica column by simple isocratic elution without any treatment of plasma. Moreover, the use of "hybrid" mixed micellar mobile phase containing anionic, non-ionic surfactants and organic solvent was effective to further enhance peak efficiency and elute highly retained hydrophobic PET radioligands. These characteristics enabled significant shorting the radiometabolite analysis procedure of PET radioligands and simplifying the experimental setup., (Copyright © 2013 Elsevier B.V. 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.)

A modular stereoselective synthesis of the vesicular monoamine transport inhibitors (+)-tetrabenazine ((+)-1) and (+)-α-dihydrotetrabenazine ((+)-2) has been developed. The approach is based on amine 4 and acid 5 as the key building blocks, which were elaborated into macrolactam 3 by amide coupling and a subsequent highly E-selective RCM reaction. Macrolactam 3 could be converted into tetrabenazine in three known steps.

A concise synthesis of tetrabenazine and dihydrotetrabenazine is described. The key feature of this synthesis is the intramolecular aza-Prins-type cyclization of an amino allylsilane via oxidative C-H activation.

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.)