A direct fixation of CO2 for isotopic labelling of hydantoins using iodine-phosphine charge transfer complexes.

Herein, we report a method for the isotopic labelling of hydantoins directly from CO2 by means of trimethyl-λ5-phosphine diiodide mediated carbonyl insertion. The method is suitable for 13C-labelling of diverse substrates and was implementated for 11C-labelling in PET-imaging facilities for the synthesis of radiotracers. Isolated yields of 90% and radiochemical yields of 89% were achieved for hydantoin containing drug candidates in formulation within 30 min with high molar activity (>400 MBq nmol-1).

Mild, Organo-Catalysed Borono-Deamination as a Key to Late-Stage Pharmaceutical Precursors and 18F-Labelled Radiotracers.

A tris(pentafluorophenyl)borane catalysed method for the synthesis of boronic acid esters from aromatic amines in yields of up to 93% was devised. Mild conditions, benign reagents, short reaction times, low temperatures and a wide substrate scope characterize the method. The reaction was found applicable to the synthesis of boronic acid ester derivatives of complex drug molecules in up to 86% isolated yield and high purity suitable for labelling. These boronates were subsequently labelled with [18F]fluoride ion in radiochemical yields of up to 55% with and even without isolation of the boronate-intermediate.

Whole-body biodistribution and radiation dosimetry of [18F]PR04.MZ: a new PET radiotracer for clinical management of patients with movement disorders.

PURPOSE: [18F]PR04.MZ is a new PET imaging agent for dopamine transporters, providing excellent image quality and allowing for the evaluation of patients with movement disorders such as Parkinson's disease. The objective of this study was to evaluate the biodistribution and radiation dosimetry of [18F]PR04.MZ by serial PET imaging. METHODS: Six healthy subjects (n = 3 males, n = 3 females) were enrolled in this study. A series of 14 whole-body PET/CT scans were acquired until 5.5 h post-injection of 200 ± 11 MBq of [18F]PR04.MZ. After rigid co-registration, volumes of interest were outlined either on CT or PET images. Time-integrated activity coefficients were calculated for selected source organs. Organ absorbed doses, and the effective dose were calculated using IDAC-Dose 2.1. RESULTS: Physiological uptake of [18F]PR04.MZ was mainly observed in the striatum, brain, liver, gall bladder, intestine, red marrow and cortical bone. [18F]PR04.MZ was primarily excreted via hepatobiliary clearance and, to a lower extent, via renal clearance. The normalized absorbed doses were highest in gall bladder wall (32.2 ± 6.4 µGy/MBq), urinary bladder wall (27.2 ± 4.5 µGy/MBq), red marrow (26.5 ± 1.4 µGy/MBq), cortical bone surface (26.3 ± 2.5 µGy/MBq), liver (22.5 ± 1.8 µGy/MBq) and kidneys (21.8 ± 1.1 µGy/MBq). The effective dose according to ICRP 60 and 103 was 16.3 ± 1.1 and 16.6 ± 1.5 µSv/MBq, respectively. CONCLUSION: [18F]PR04.MZ has a favourable dosimetry profile, comparable to those of other 18F-labelled PET tracers, and is suitable for larger clinical applications. Trial registration CEC SSM Oriente, Santiago, Chile, permit 20140520.

A General Protocol for Cu-Mediated Fluoro-deamination: Sandmeyer Fluorination of Diverse Aromatic Substrates.

A Cu(I)-mediated fluoro-deamination method for nucleophilic radiofluorination was devised. The method affords fluorinated aromatic products directly from anilines under both no-carrier added and stoichiometric conditions. Isolated radiochemical yields range from 11% to 81% with high radiochemical purities and a molar activity of 58 MBq/nmol. The reaction conditions were implemented successfully in an automated process for production of (S)-4[18F]fluorogluthetimide on a radiosynthesis module.

[18F]PR04.MZ PET/CT Imaging for Evaluation of Nigrostriatal Neuron Integrity in Patients With Parkinson Disease.

INTRODUCTION: Degeneration of dopaminergic, nigrostriatal neurons is the hallmark of Parkinson disease (PD), and PET quantification of dopamine transporters is a widely accepted method for differential diagnosis between idiopathic PD and essential tremor. [18F]PR04.MZ is a new PET tracer with excellent imaging properties allowing for precise quantification of striatal and extrastriatal dopamine transporter. Here we describe our initial experience with [18F]PR04.MZ PET/CT in a larger cohort of healthy controls and PD patients as a proof-of-concept study for this tracer. METHODS: Eighteen healthy subjects, 19 early PD patients (Hoehn-Yahr I-II), and 13 moderate-advanced PD patients (Hoehn-Yahr III-IV) underwent static PET/CT scans 60 to 90 minutes after injection of 5.16 ± 1.03 mCi (191 ± 38 MBq) [18F]PR04.MZ. Specific binding ratios (SBRs) were calculated for caudate nucleus, anterior putamen, posterior putamen, substantia nigra (SNpc), compared between different groups and correlated with clinical ratings. RESULTS: [18F]PR04.MZ showed very high and specific uptake in the putamen, caudate, and substantia nigra pars compacta and very low nonspecific binding in other brain regions, and SBR values for the control group were 22.3 ± 4.1, 19.1 ± 3.5, and 5.4 ± 1.2, respectively. A reduction of SBR values was observed in all regions and in both initial and moderate PD, ranging from 35% to 89% (P < 0.001). The observed pattern of reduction was posterior putamen > anterior putamen > substantia nigra pars compacta > caudate, with contralateral posterior putamen being the most affected region. Rostrocaudal depletion gradient was evident in all PD patients and progression correlated with motor manifestations. CONCLUSIONS: [18F]PR04.MZ PET/CT is a highly sensitive imaging modality for the detection of dopaminergic deficit in nigrostriatal pathways in PD.

Pharmacological Characterization of Low-to-Moderate Affinity Opioid Receptor Agonists and Brain Imaging with 18F-Labeled Derivatives in Rats.

The 3,4-dichloro-N-(1-(dimethylamino)cyclohexyl)methyl benzamide scaffold was studied as a template for 18F-positron emission tomography (18F-PET) radiotracer development emphasizing sensitivity to changes in opioid receptor (OR) occupancy over high affinity. Agonist potency, binding affinity, and relevant pharmacological parameters of 15 candidates were investigated. Two promising compounds 3b and 3e with µ-OR (MOR) selective agonist activity in the moderate range (EC50 = 1-100 nM) were subjected to 18F-fluorination, autoradiography, and small-animal PET imaging. Radioligands [18F]3b and [18F]3e were obtained in activity yields of 21 ± 5 and 23 ± 4% and molar activities of 25-40 and 200-300 GBq/µmol, respectively. Displaceable binding matching MOR distribution in the brain was confirmed by imaging. Radioligands showed a rapid pharmacokinetic profile; however, metabolite-corrected, blood-based modeling was required for data analysis. Observed BPND was low, although treatment with naloxone leads to a marked decrease in specific binding, confirming the discovery of a new template for 18F-labeled OR-agonist PET ligands.

Discovery of a Lead Brain-Penetrating Gonadotropin-Releasing Hormone Receptor Antagonist with Saturable Binding in Brain.

We report the synthesis, radiosynthesis and biological characterisation of two gonadotropin-releasing hormone receptor (GnRH-R) antagonists with nanomolar binding affinity. A small library of GnRH-R antagonists was synthesised in 20-67 % overall yield with the aim of identifying a high-affinity antagonist capable of crossing the blood-brain barrier. Binding affinity to rat GnRH-R was determined by autoradiography in competitive-binding studies against [125 I]buserelin, and inhibition constants were calculated by using the Cheng-Prusoff equation. The radioligands were obtained in 46-79 % radiochemical yield and >95 % purity and with a molar activity of 19-38 MBq/nmol by direct nucleophilic radiofluorination. Positron emission tomography imaging in rat under baseline conditions in comparison to pretreatment with a receptor-saturating dose of GnRH antagonist revealed saturable uptake (0.1 %ID/mL) into the brain.

Evaluation of [18F]-N-Methyl lansoprazole as a Tau PET Imaging Agent in First-in-Human Studies.

Development of positron emission tomography (PET) imaging agents capable of quantifying tau aggregates in neurodegenerative disorders such as Alzheimer's disease (AD) is of enormous importance in the field of dementia research. The aim of the present study was to conduct first-in-man imaging studies with the potential novel tau imaging agent [18F]N-methyl lansoprazole ([18F]NML). Herein we report validation of the synthesis of [18F]NML for clinical use by labeling the trifluoromethyl group via radiofluorination of the corresponding gem-difluoro enol ether precursor. This is the first use of this method for clinical production of PET radiotracers and confirmed that it can be readily implemented at multiple production facilities to provide [18F]NML in good noncorrected radiochemical yield (3.4 ± 1.5 GBq, 4.6% ± 2.6%) and molar activity (120.1 ± 186.3 GBq/µmol), excellent radiochemical purity (>97%), and suitable for human use (n = 15). With [18F]NML in hand, we conducted rodent biodistribution, estimates of human dosimetry, and preliminary evaluation of [18F]NML in human subjects at two imaging sites. Healthy controls (n = 4) and mildly cognitively impaired (MCI) AD patients (n = 6) received [18F]NML (tau), [18F]AV1451 (tau), and [18F]florbetaben or [18F]florbetapir (amyloid) PET scans. A single progressive supranuclear palsy (PSP) patient also received [18F]NML and [18F]AV1451 PET scans. [18F]NML showed good brain uptake, reasonable pharmacokinetics, and appropriate imaging characteristics in healthy controls. The mean ± SD of the administered mass of [18F/19F]NML was 2.01 ± 2.17 µg (range, 0.16-8.27 µg) and the mean administered activity was 350 ± 62 MBq (range, 199-403 MBq). There were no adverse or clinically detectable pharmacologic effects in any of the 11 subjects, and no significant changes in vital signs were observed. However, despite high affinity for tau in vitro, brain retention in MCI/AD and PSP patients was low, and there was no evidence of specific signals in vivo that corresponded to tau. Although it is still unclear why clinical translation of the radiotracer was unsuccessful, we nevertheless conclude that further development of [18F]NML as a tau PET imaging agent is not warranted at this time.

Evaluation by metabolic profiling and in vitro autoradiography of two promising GnRH-receptor ligands for brain SPECT imaging.

The increased expression of gonadotropin releasing hormone receptor (GnRH-R) in brain has been strongly linked to Alzheimer disease. Therefore, the development of radiolabeled imaging agents for GnRH-R is relevant for early diagnosis of Alzheimer disease. We have recently disclosed the discovery of two promising compounds displaying nanomolar-range affinity for the GnRH-R. In the present study, a preclinical evaluation of the compound properties was performed to evaluate their potential as single photon emission computed tomography (SPECT) radiotracers for imaging the GnRH-receptor. The compounds were assessed in vitro by performing serum stability analysis by human and rat serum, metabolic profiling by human liver microsomes, and exploratory rat brain autoradiography. The investigated compounds displayed satisfactory stability against human, rat serum, and liver microsomal metabolism, which favors their potential as SPECT-imaging agents. Additionally, we identified and quantified the formation rate of the metabolites by fragmentation of up to five mass spectrometric stages. The GnRH-R rat brain specificity of these compounds was tested in competition with a known ligand for the receptor and the in vitro autoradiography confirmed that compounds 3 and 4 binds to rat GnRH-R in different rat brain regions.

Pharmacokinetic evaluation of [18F]PR04.MZ for PET/CT imaging and quantification of dopamine transporters in the human brain.

PURPOSE: Dopamine transporters (DAT) modulate pre-synaptic dopamine and physiological functions such as movement and reward. DAT also mirrors disease state in neurological disorders, rendering it an essential diagnostic target. [18F]PR04.MZ is a new PET imaging agent for DAT with an improved affinity and selectivity profile, for which we here describe the complete pharmacokinetic evaluation in healthy controls. METHODS: Thirty-two healthy subjects underwent T1-weighted MRI and dynamic PET scans for 180 min with arterial blood sampling (n = 5) or 90 min without blood sampling (n = 25) after injection of 197.6 ± 12.2 MBq [18F]PR04.MZ. Blood and plasma metabolite analysis were performed. MRI-based normalization of brain images, delineation of VOIs, and kinetic modeling was conducted to determine distribution volumes (Vt) and binding potentials (BPnd). The impact of scan duration was evaluated and repeated PET scans were performed to assess test-retest variability (n = 5). A static imaging protocol has been validated for clinical applications. RESULTS: [18F]PR04.MZ showed rapid metabolization in circulation, very high uptake in striatum and midbrain, and very low non-specific binding. The two-tissue compartment model 2TCM provided best fits for measured time-activity-curves and calculated Vts in putamen, caudate, substantia nigra pars compacta (SNpc), and cerebellar cortex were 11.83, 9.73, 2.12, and 0.57, respectively. All non-invasive models correlated well with BPnd values derived from 2TCM but underestimated DAT availability by about 28-33%. Of those, simplified reference tissue model (SRTM) provided the best fits, lowest Akaike Information Criteria values, and BPnd values of 14.82, 11.95, and 2.63 in putamen, caudate, and SNpc, respectively. BPnd estimates for striatal regions and SNpc were stable between 90 and 130 min post-injection. Test-retest results were excellent, showing low variability in all and excellent reliability in most relevant regions. Static imaging from 60 to 90-min post-injection is a viable alternative for quantification. CONCLUSIONS: [18F]PR04.MZ is a PET tracer with very high affinity, selectivity, and specific uptake in striatum and midbrain. 2TCM and SRTM provide good fits, high and stable Vts or BPnds, and good test-retest reliability for precise quantification of DAT in human subjects.

Synthesis, radiosynthesis, and positron emission tomography neuroimaging using 5-[18 F]fluoro-L-amino suberate.

System xc- (Sxc -) has emerged as a new biological target for PET studies to detect oxidative and excitotoxic stress. Notably, applications have, thus far, been limited to tumour imaging although Sxc- ) may play a major role in neurodegeneration. The synthesis procedures of tosylate precursor and its translation to Sxc - PET tracer 5[18F]fluoro-L-amino suberate by manual and automated radiosyntheses are described. A brain-PET study has been conducted to evaluate the tracer uptake into brain in healthy mice.

Synthesis and Characterization in Rodent Brain of the Subtype-Selective NR2B NMDA Receptor Ligand [11C]Ro04-5595 as a Potential Radiotracer for Positron Emission Tomography.

The NR2B subunit of the N-methyl-d-aspartate (NMDA) receptor has been implicated in controlling synaptic plasticity, memory, and learning. Herein, we describe an 11C-labeled PET radiotracer based on 1-(4-chlorophenethyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol, Ro04-5595. The radiotracer was evaluated in rats using PET. The PET study showed a good pharmacokinetic profile with rapid uptake and washout over 90 min. Complementary high-resolution autoradiographic images using [3H]Ro04-5595 demonstrated strong binding in NR2B receptor-rich regions and low binding in cerebellum where NR2B concentration is low. We conclude to have developed a selective NR2B receptor radioligand suitable for quantitative and qualitative imaging of a NR2B receptor distribution in vitro and in vivo.

Insula serotonin 2A receptor binding and gene expression contribute to serotonin transporter polymorphism anxious phenotype in primates.

Genetic variation in the serotonin transporter gene (SLC6A4) is associated with vulnerability to affective disorders and pharmacotherapy efficacy. We recently identified sequence polymorphisms in the common marmoset SLC6A4 repeat region (AC/C/G and CT/T/C) associated with individual differences in anxiety-like trait, gene expression, and response to antidepressants. The mechanisms underlying the effects of these polymorphisms are unknown, but a key mediator of serotonin action is the serotonin 2A receptor (5HT2A). Thus, we correlated 5HT2A binding potential (BP) and RNA gene expression in 16 SLC6A4 genotyped marmosets with responsivity to 5HT2A antagonism during the human intruder test of anxiety. Voxel-based analysis and RNA measurements showed a reduction in 5HT2A BP and gene expression specifically in the right posterior insula of individuals homozygous for the anxiety-related variant AC/C/G. These same marmosets displayed an anxiogenic, dose-dependent response to the human intruder after 5HT2A pharmacological antagonism, while CT/T/C individuals showed no effect. A voxel-based correlation analysis, independent of SLC6A4 genotype, revealed that 5HT2A BP in the adjacent right anterior insula and insula proisocortex was negatively correlated with trait anxiety scores. Moreover, 5HT2A BP in both regions was a good predictor of the size and direction of the acute emotional response to the human intruder threat after 5HT2A antagonism. Our findings suggest that genetic variation in the SLC6A4 repeat region may contribute to the trait anxious phenotype via neurochemical changes in brain areas implicated in interoceptive and emotional processing, with a critical role for the right insula 5HT2A in the regulation of affective responses to threat.

Influx rate of 18F-fluoroaminosuberic acid reflects cystine/glutamate antiporter expression in tumour xenografts.

PURPOSE: 18F-fluoroaminosuberic acid (18F-FASu) is a recently developed amino acid tracer for positron emission tomography (PET) of oxidative stress that may offer improved tumour assessment over the conventional tracer 18F-fluorodeoxyglucose (18F-FDG). Our aim was to evaluate and relate dynamic 18F-FASu and 18F-FDG uptake with pharmacokinetic modelling to transporter protein expression levels in a panel of diverse tumour xenograft lines. METHODS: Four different tumour xenograft lines were implanted in female athymic nude mice: MAS98.12 and HBCx3 (breast), TPMX (osteosarcoma) and A549 (lung). Dynamic PET over 60 min was performed on a small animal unit. The time-activity curves (TACs) for 18F-FASu and 18F-FDG in individual tumours were used to extract early (SUVE; 2 min p.i.) and late (SUVL; 55 min p.i.) standardised uptake values. Pharmacokinetic two-tissue compartment models were applied to the TACs to estimate rate constants K1-k4 and blood volume fraction vB. Relative levels of cystine/glutamate antiporter subunit xCT were assessed by western blotting, and expression of GLUT1 and CD31 by immunohistochemistry. RESULTS: 18F-FASu showed higher SUVE, whilst 18F-FDG exhibited higher SUVL. Influx rate K1 for 18F-FASu was significantly correlated with xCT levels (p = 0.001) and was significantly higher than K1 for 18F-FDG (p < 0.001). K1 for 18F-FDG was significantly correlated with GLUT1 levels (p = 0.002). vB estimated from 18F-FASu and 18F-FDG TACs was highly consistent and significantly correlated (r = 0.85, p < 0.001). Two qualitatively different 18F-FASu uptake profiles were identified: type α with low xCT expression and low K1 (A549 and HBCx3), and type ß with high xCT expression and high K1 (MAS98.12 and TPMX). CONCLUSION: The influx rate of 18F-FASu reflects xCT activity in tumour xenografts. Dynamic PET with pharmacokinetic modelling is needed to fully appraise 18F-FASu distribution routes.

First in vivo evaluation of a potential SPECT brain radiotracer for the gonadotropin releasing hormone receptor.

OBJECTIVES: In vivo evaluations of a gonadotropin releasing hormone-receptor single photon emission computed tomography radiotracer for non-invasive detection of gonadotropin releasing homone-receptors in brain. RESULTS: We have used a simple, robust and high-yielding procedure to radiolabel an alpha-halogenated bioactive compound with high radiochemical yield. Literature findings showed similar alpha-halogenated compounds suitable for in vivo evaluations. The compound was found to possess nano molar affinity for the gonadotropin releasing hormone-receptor in a competition dependent inhibition study. Furthermore, liquid chromatography-mass spectrometry analysis in saline, human and rat serum resulted in 46%, 52% and 44% stability after incubation for 1 h respectively. In addition, rat brain single photon emission computed tomography and biodistribution studies gave further insight into the nature of the compound as a radiotracer.

Image-Guided Development of Heterocyclic Sulfoxides as Ligands for Tau Neurofibrillary Tangles: From First-in-Man to Second-Generation Ligands.

Positron emission tomography (PET) imaging of misfolded protein aggregates that form in neurodegenerative processes of the brain is key to providing a robust marker for improved diagnosis and evaluation of treatments. We report the development of advanced radiotracer candidates based on the sulfoxide scaffold found in proton pump inhibitors (lansoprazole, prevacid) with inherent affinity to neurofibrillary tangles in Alzheimer's disease and related disorders (e.g., dementia with Lewy bodies and the frontotemporal degeneration syndrome). First-in-man results obtained with [18F]lansoprazole and N-methyl-[18F]lansoprazole were used to guide the design of a set of 24 novel molecules with suitable properties for neuroimaging with PET. Compounds were synthesized and characterized pharmacologically, and the binding affinity of the compounds to synthetic human tau-441 fibrils was determined. Selectivity of binding was assessed using α-synuclein and ß-amyloid fibrils to address the key misfolded proteins of relevance in dementia. To complete the pharmacokinetic profiling in vitro, plasma protein binding and lipophilicity were investigated. Highly potent and selective new radiotracer candidates were identified for further study.

Stereospecific radiosynthesis of 3-fluoro amino acids: access to enantiomerically pure radioligands for positron emission tomography.

A variety of substituted non-racemic aziridine-2-carboxylates equivalent to amino acids were prepared and subjected to ring opening reaction by [18F/19F]fluoride. The regio and stereospecific ring opening depends on the substituents on the nitrogen as well as both the carbons of aziridines. The applicability of the methods to afford access to 3-[18F/19F]fluoro amino acids are illustrated.

Poly(ADP-ribose) Polymerase in Neurodegeneration: Radiosynthesis and Radioligand Binding in ARC-SWE tg Mice.

We report the synthesis, radiosynthesis, and characterization of a radioligand for poly(ADP-ribose) polymerase (PARP). PARP is of central importance in cell homeostasis, neuroplasticity, and neurodegeneration in the brain. A radiolabeled PARP inhibitor was developed and used for autoradiographic quantification of PARP protein concentration in wild-type and transgenic rodent brains ex vivo in high resolution. The binding of [3H]rucaparib was found to be confined to PARP-expressing domains, for example, cerebellar cortex or hippocampal regions in both models. Saturation binding experiments confirmed selective and reversible binding to a single site ( Kd = 1.1 ± 0.2 nM).

FDG-based quantitative comparison of glucose metabolism in vitro, exemplified by a head-to-head comparison between a triple-negative breast cancer cell line and a non-malignant foetal cell line.

BACKGROUND: Glucose metabolism can be studied in vitro by a variety of means, also by fluorodeoxyglucose (FDG). As an example of the potential use we have compared the high glucose consumption in cancer cells and in transformed non-malignant foetal cells. The high glucose metabolism in cancer cells is not primarily for the production of energy, a large proportion is transformed to lactate only, producing two instead of potentially 32 ATP equivalents. The secreted lactate reduces the pH in the local microenvironment and gives malignant cells, more apt to thrive in hypoxic and acid environments, a competitive advantage. PURPOSE: To demonstrate the use of FDG in head-to-head comparison of glucose uptake and lactate production in a highly malignant and a highly proliferative non-malignant cell line. MATERIALS AND METHODS: Cell cultures of a foetal kidney cell line and a triple negative breast cancer cell line were incubated with FDG for one hour, washed, harvested and the radioactivity content in the cells was measured in a well counter. The lactate concentration was measured in conditioned medium. RESULTS: The FDG uptake was consistently higher in the non-malignant foetal cell line. The levels of lactate in the conditioned medium were similar. CONCLUSION: Quantitative comparison of glucose metabolism by in vitro use of FDG is a practical, cheap and rapid method. Some foetal cells have higher glucose uptake and produce as much lactate as this highly malignant cell line. This is an argument against the high glucose consumption being related solely to malignancy.