Beta Cell Imaging-From Pre-Clinical Validation to First in Man Testing.

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.

Deutetrabenazine: Treatment of hyperkinetic aspects of Huntington's disease, tardive dyskinesia and Tourette syndrome.

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.

Cardiac Amyloid Imaging with 18F-Florbetaben PET: A Pilot Study.

Our aim was to determine the feasibility of 18F-florbetaben PET in diagnosing cardiac amyloidosis. METHODS: 18F-florbetaben PET was performed on 14 patients: 5 amyloid light chain, 5 amyloid transthyretin, and 4 control with hypertensive heart disease. Qualitative and quantitative assessments of 18F-florbetaben activity were performed using the SUVmean of the left ventricular myocardium and blood pool and calculation of target-to-background SUV ratio. Myocardial 18F-forbetaben retention was also calculated as the percentage mean myocardial SUV change between 0 and 5 min and 15 and 20 min after radiotracer injection. Global left ventricular longitudinal and right ventricular free wall longitudinal strain were calculated using 2-dimensional speckle-tracking echocardiography. RESULTS: Target-to-background SUV ratio and percentage myocardial 18F-forbetaben retention were higher in amyloid patients than in hypertensive controls. A cutoff of 40% was able to differentiate between cardiac amyloid patients and hypertensive controls. Percentage myocardial 18F-forbetaben retention was an independent determinant of both global left ventricular longitudinal and right ventricular free wall longitudinal strain via an inverse curve relationship. CONCLUSION: 18F-florbetaben PET imaging can accurately identify and differentiate between cardiac amyloidosis and hypertensive heart disease. Percentage myocardial 18F-florbetaben retention was an independent determinant of myocardial dysfunction in cardiac amyloidosis.

N-terminus regulation of VMAT2 mediates methamphetamine-stimulated efflux.

The 20 amino acid (AA) N-terminus of the vesicular monoamine transporter 2 (VMAT2) was examined as a regulator of VMAT2 function. Removal of the first 16 or 19 AAs of the N-terminus resulted in a molecule with reduced ability to sequester [(3)H]-5HT. A glutathione-S-transferase-construct of the N-terminus underwent phosphorylation in the presence of PKC at serines 15 and 18. These putative phosphorylation sites were examined for effects on function. Phospho-mimetic substitution of serines 15 and 18 with aspartate in the full-length VMAT2 resulted in reduced [(3)H]-5HT sequestration and reduced methamphetamine (METH)-stimulated efflux of preloaded [(3)H]-5HT. In contrast, mutation of serines 15 and 18 to alanines maintained intact net substrate sequestration but eliminated METH-stimulated efflux of pre-accumulated [(3)H]-5HT. In summary, these data suggest a model in which the VMAT2 N-terminus regulates monoamine sequestration.

Direct intranigral administration of an ubiquitin proteasome system inhibitor in rat: behavior, positron emission tomography, immunohistochemistry.

Several independent lines of research suggest that disruption of the ubiquitin proteasome system (UPS) may play a role in the pathophysiology of Parkinson's disease. Direct intracerebral injection of UPS inhibitors (e.g. lactacystin) in animals has consistently produced important features of the disease. In this study, a range of lactacystin doses (0.5, 1, 2, 10 and 20 µg) were injected into the right substantia nigra in rats to determine the ideal dose required to produce a robust and specific lesion of the dopamine nigro-striatal system and motor deficits. Motor behavior, assessed with the tapered ledged beam task, was severely affected in animals that received high doses (10 and 20 µg) but only mild, impairments were observed in animals that received low doses (0.5, 1, and 2 µg). Positron emission tomography was performed with a dedicated small animal scanner on the rats following the injection of the radio-labeled tracer (±)[(11)C]dihydrotetrabenazine (DTBZ) which labels vesicular monoamine transporter type 2. Severe loss of [(11)C]DTBZ binding in the ipsilateral striatum was observed in the higher dose groups and mild loss was observed in the low dose groups. Stereological cell counting of tyrosine hydroxylase immunoreactive cells in the substantia nigra and the ventral tegmental area indicated a dose dependent loss of dopaminergic neurons. Significant correlations were found between the behavioral motor deficits, striatal [(11)C]DTBZ binding and cell counts of tyrosine hydroxylase immunoreactive cells. Taken together these results indicate that intranigral injection of lactacystin produces dose dependent effects on the dopamine nigro-striatal system and a dose of 10 µg will produce a consistent severe lesion.

Sex differences in methamphetamine toxicity in mice: effect on brain dopamine signaling pathways.

Male mice were reported to display greater methamphetamine-induced neurotoxicity than females. The present study evaluated the involvement of phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK1/2) pathways in this sex-dependent methamphetamine toxicity. Intact female and male mice were administered methamphetamine (20 or 40mg/kg) and euthanized a week later. Dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) autoradiography in the lateral striatum showed a greater sensitivity in male mice treated with 20mg/kg methamphetamine compared to female mice. Striatal dopamine concentration and DAT autoradiography showed a more extensive depletion in male mice given 40mg/kg methamphetamine compared to female mice. Mice administered 40mg/kg methamphetamine showed no sex difference in striatal VMAT2 autoradiography. In the substantia nigra, DAT specific binding was decreased only in male mice treated with 40mg/kg methamphetamine and DAT mRNA levels decreased in methamphetamine-treated female and male mice. Methamphetamine-treated male mice presented a dose-dependent decrease of VMAT2 mRNA levels. Methamphetamine reduced insulin-like growth factor 1 receptor levels in females at both methamphetamine doses tested whereas it elevated G protein-coupled estrogen receptor 1 (GPER1) only in male mice. Phosphorylated Akt levels decreased only in male mice treated with 40mg/kg methamphetamine. Glycogen synthase kinase 3ß levels were reduced in male mice at both methamphetamine doses tested and in females receiving 40mg/kg. Bcl-2 levels were increased in male mice treated with methamphetamine, whereas ERK1/2 and BAD levels were unchanged. These results implicate some of the signaling pathways associated with the sex differences in methamphetamine-induced toxicity.

Whole-body biodistribution and radiation dosimetry of 18F-FP-(+)-DTBZ (18F-AV-133): a novel vesicular monoamine transporter 2 imaging agent.

UNLABELLED: Vesicular monoamine transporter 2 (VMAT2) is highly expressed in the endocrine cells and brain. We investigated the biodistribution and radiation dosimetry of (2R,3R,11bR)-9-(3-(18)F-fluoropropoxy)-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol ((18)F-FP-(+)-dihydrotetrabenazine [DTBZ] or (18)F-AV-133), a potential VMAT2 imaging agent showing encouraging results in humans, to facilitate its future clinical use. METHODS: Nine healthy human subjects (mean age +/- SD, 58.6 +/- 4.2 y) were enrolled for the whole-body PET scan. Serial images were acquired for 3 h immediately after a bolus injection of 390.7 +/- 22.9 MBq of (18)F-AV-133 per individual. The source organs were delineated on PET/CT images. The OLINDA/EXM application was used to determine the equivalent dose for individual organs. RESULTS: The radiotracer did not show any noticeable adverse effects for the 9 subjects examined. The radioactivity uptake in the brain was the highest at 7.5% +/- 0.6% injected dose at 10 min after injection. High absorbed doses were found in the pancreas, liver, and upper large intestine wall. The highest-dosed organ, which received 153.3 +/- 23.8 microGy/MBq, was the pancreas. The effective dose equivalent and effective dose for (18)F-AV-133 were 36.5 +/- 2.8 and 27.8 +/- 2.5 microSv/MBq, respectively. These values are comparable to those reported for any other (18)F-labeled radiopharmaceutical. CONCLUSION: (18)F-AV-133 is safe, with appropriate biodistribution and radiation dosimetry for imaging VMAT2 sites in humans.

Whole body [11C]-dihydrotetrabenazine imaging of baboons: biodistribution and human radiation dosimetry estimates.

PURPOSE: Vesicular monoamine transporter type 2 abundance quantified using the radiotracer [(11)C]-dihydrotetrabenazine (DTBZ) has been used to study diagnosis and pathogenesis of dementia and psychiatric disorders in humans. In addition, it may be a surrogate marker for insulin-producing pancreatic beta cell mass, useful for longitudinal measurements using positron emission tomography to track progression of autoimmune diabetes. To support the feasibility of long-term repeated administrations, we estimate the biodistribution and dosimetry of [(11)C]-DTBZ in humans. METHODS: Five baboon studies were acquired using a Siemens ECAT camera. After transmission scanning, 165-210 MBq of [(11)C]-DTBZ were injected, and dynamic whole body emission scans were conducted. Time-activity data were used to obtain residence times and estimate absorbed radiation dose according to the MIRD model. RESULTS: Most of the injected tracer localized to the liver and the lungs, followed by the intestines, brain, and kidneys. The highest estimated absorbed radiation dose was in the stomach wall. CONCLUSIONS: The largest radiation dose from [(11)C]-DTBZ is to the stomach wall. This dose estimate, as well as the radiation dose to other radiosensitive organs, must be considered in evaluating the risks of multiple administrations.

In vivo measurement of density and affinity of the monoamine vesicular transporter in a unilateral 6-hydroxydopamine rat model of PD.

This is the first in vivo determination of the vesicular monoamine transporter (VMAT2) density (B(max)) and ligand-transporter affinity (K(d)(app)) in six unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats using micro-positron emission tomography (PET) imaging with [(11)C]-(+)-alpha-dihydrotetrabenazine (DTBZ). A multiple ligand concentration transporter assay (MLCTA) was used to determine a B(max) value of 178+/-32 pmol/mL and a K(d)(app) of 47.7+/-9.3 pmol/mL for the non-lesioned side and 30.52+/-5.84 and 43.4+/-15.52 pmol/mL for the lesioned side, respectively. While B(max) was significantly different between the two sides, no significant difference was observed for the K(d)(app). In addition to demonstrating the feasibility of in vivo Scatchard analysis in rats, these data confirm the expectation that a 6-OHDA lesion does not affect the affinity; a much simpler binding potential (BP) measure can thus be used as a marker of lesion severity (LS) in this rat model of Parkinson's disease. A transporter occupancy curve demonstrated negligible transporter occupancy ( approximately 1%) at a specific activity (SA) of 1100 nCi/pmol (assuming an injected dose of 100 microCi/100 g), while 10% occupancy was estimated at 100 nCi/pmol. An indirect measurement indicated that the degree of occupancy as a function of SA is independent of LS. Finally, BP measurement reproducibility was assessed and found to be 11%+/-7% for the healthy and 8%+/-12% for the lesioned side. Quantitative PET results can thus be obtained even for severely lesioned animals with the striatum on one side not clearly visible provided accurate image analysis methods are used.

Methylphenidate redistributes vesicular monoamine transporter-2: role of dopamine receptors.

It is well accepted that methylphenidate (MPD) inhibits dopamine (DA) transporter function. In addition to this effect, this study demonstrates that MPD increases vesicular [3H]DA uptake and binding of the vesicular monoamine transporter-2 (VMAT-2) ligand dihydrotetrabenazine (DHTBZ) in a dose- and time-dependent manner in purified striatal vesicles prepared from treated rats. This change did not result from residual MPD introduced by the original in vivo treatment, because application of MPD in vitro (< or =1 miccrom) was without effect, and higher concentrations decreased vesicular [3H]DA uptake. In addition, MPD treatment increased and decreased VMAT-2 immunoreactivity in striatal vesicle subcellular and plasmalemmal membrane fractions, respectively. The MPD-induced increase in both VMAT-2 immunoreactivity and DHTBZ binding was attenuated by pretreatment in vivo with either the DA D(1) receptor antagonist SCH23390 or the DA D2 receptor antagonist eticlopride. Coadministration of these antagonists in vivo inhibited completely the MPD-induced increase in DHTBZ binding in the purified vesicular preparation. These observations suggest a role for DA in the MPD-induced redistribution of VMAT-2. The implications of this phenomenon will be discussed.

Lobeline displaces [3H]dihydrotetrabenazine binding and releases [3H]dopamine from rat striatal synaptic vesicles: comparison with d-amphetamine.

Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d-amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobeline's action and compare its effects with those of d-amphetamine. [3H]Dihydrotetrabenazine ([3H]DTBZ), used routinely to probe a high-affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a KD of 1.67 nM and Bmax of 8.68 pmol/mg of protein. Lobeline inhibited [3H]DTBZ binding with an IC50 of 0.90 microM, consistent with its previously reported IC50 of 0.88 microM for inhibition of [3H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d-amphetamine inhibited [3H]DTBZ binding to vesicle membranes with an IC50 of 39.4 microM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d-amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [3H]DA release from [3H]DA-preloaded synaptic vesicles in the absence of drug revealed a t1/2 of 2.12 min. Lobeline and d-amphetamine evoked [3H]DA release with EC50 values of 25.3 and 2.22 microM, respectively. At a concentration 10 times the EC50, lobeline and d-amphetamine significantly decreased the t1/2 of [3H]DA release to 1.58 and 1.48 min, respectively. Thus, in contrast to d-amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28-fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.