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Purpose To provide a comprehensive assessment of the novel 18 kDa translocator protein (TSPO) radiotracer, [18F]LW223, kinetics in the heart and brain when using a simplified imaging approach. Methods Naive adult rats and rats with surgically induced permanent coronary artery ligation received a bolus intravenous injection of [18F]LW223 followed by 120 min PET scanning with arterial blood sampling throughout. Kinetic modelling of PET data was applied to estimated rate constants, total volume of distribution (VT) and binding potential transfer corrected (BPTC) using arterial or image-derived input function (IDIF). Quantitative bias of simplified protocols using IDIF versus arterial input function (AIF) and stability of kinetic parameters for PET imaging data of different length (40–120 min) were estimated. Results PET outcome measures estimated using IDIF significantly correlated with those derived with invasive AIF, albeit with an inherent systematic bias. Truncation of the dynamic PET scan duration to less than 100 min reduced the stability of the kinetic modelling outputs. Quantification of [18F]LW223 uptake kinetics in the brain and heart required the use of different outcome measures, with BPTC more stable in the heart and VT more stable in the brain. Conclusion Modelling of [18F]LW223 PET showed the use of simplified IDIF is acceptable in the rat and the minimum scan duration for quantification of TSPO expression in rats using kinetic modelling with this radiotracer is 100 min. Carefully assessing kinetic outcome measures when conducting a systems level as oppose to single-organ centric analyses is crucial. This should be taken into account when assessing the emerging role of the TSPO heart-brain axis in the field of PET imaging.

Visual Abstract, Myocardial infarction (MI) is one of the leading causes of death worldwide, and inflammation is central to tissue response and patient outcomes. The 18-kDa translocator protein (TSPO) has been used in PET as an inflammatory biomarker. The aims of this study were to screen novel, fluorinated, TSPO radiotracers for susceptibility to the rs6971 genetic polymorphism using in vitro competition binding assays in human brain and heart; assess whether the in vivo characteristics of our lead radiotracer, 18F-LW223, are suitable for clinical translation; and validate whether 18F-LW223 can detect macrophage-driven inflammation in a rat MI model. Methods: Fifty-one human brain and 29 human heart tissue samples were screened for the rs6971 polymorphism. Competition binding assays were conducted with 3H-PK11195 and the following ligands: PK11195, PBR28, and our novel compounds (AB5186 and LW223). Naïve rats and mice were used for in vivo PET kinetic studies, radiometabolite studies, and dosimetry experiments. Rats underwent permanent coronary artery ligation and were scanned using PET/CT with an invasive input function at 7 d after MI. For quantification of PET signal in the hypoperfused myocardium, K1 (rate constant for transfer from arterial plasma to tissues) was used as a surrogate marker of perfusion to correct the binding potential for impaired radiotracer transfer from plasma to tissue (BPTC). Results: LW223 binding to TSPO was not susceptible to the rs6971 genetic polymorphism in human brain and heart samples. In rodents, 18F-LW223 displayed a specific uptake consistent with TSPO expression, a slow metabolism in blood (69% of parent at 120 min), a high plasma free fraction of 38.5%, and a suitable dosimetry profile (effective dose of 20.5–24.5 μSv/MBq). 18F-LW223 BPTC was significantly higher in the MI cohort within the infarct territory of the anterior wall relative to the anterior wall of naïve animals (32.7 ± 5.0 vs. 10.0 ± 2.4 cm3/mL/min, P ≤ 0.001). Ex vivo immunofluorescent staining for TSPO and CD68 (macrophage marker) resulted in the same pattern seen with in vivo BPTC analysis. Conclusion: 18F-LW223 is not susceptible to the rs6971 genetic polymorphism in in vitro assays, has favorable in vivo characteristics, and is able to accurately map macrophage-driven inflammation after MI.

The authors are grateful to EPSRC (EP/R511705/1 and a studentship to K.M.O., EP/K503058/1) and the University of Glasgow. We thank the University of St Andrews (M.J.W) and CAPES/Science Without Borders (C.P.F) for PhD studentships. An investigation into the mechanism of Cu-catalyzed aryl boronic acid halodeboronation using electrophilic halogen reagents is reported. Evidence is provided to show that this takes place via a boronate-driven ipso-substitution pathway and that Cu is not required for these processes to operate: general Lewis base catalysis is operational. This in turn allows the rational development of a general, simple, and effective base-catalyzed halodeboronation that is amenable to the preparation of 125I-labeled products for SPECT applications. Publisher PDF

Background Prostate cancer is highly prevalent worldwide. Androgen deprivation therapy (ADT) remains the treatment of choice for incurable prostate cancer, but majority of patients develop disease recurrence following ADT. There is therefore an urgent need for early detection of treatment resistance. Methods Isogenic androgen-responsive (CWR22Res) and castration-resistant (22Rv1) human prostate cancer cells were implanted into the anterior lobes of the prostate in CD-1 Nu mice to generate prostate orthografts. Castrated mice bearing CWR22Res and 22Rv1 orthografts mimic clinical prostate cancer following acute and chronic ADT, respectively. 18F-Fluciclovine (1-amino-3-fluorocyclobutane-1-carboxylic acid) with a radiochemical purity of > 99% was produced on a FASTlab synthesiser. Ki67 staining in endpoint orthografts was studied. Western blot, quantitative RT-PCR and next-generation sequencing transcriptomic analyses were performed to assess the expression levels of amino acid transporters (including LAT1 and ASCT2, which have been implicated for Fluciclovine uptake). Longitudinal metabolic imaging with 18F-Fluciclovine-based positron emission tomography (PET) was performed to study tumour response following acute and chronic ADT. Results Both immunohistochemistry analysis of endpoint prostate tumours and longitudinal 18F-Fluciclovine imaging revealed tumour heterogeneity, particularly following ADT, with in vivo 18F-Fluciclovine uptake correlating to viable cancer cells in both androgen-proficient and castrated environment. Highlighting tumour subpopulation following ADT, both SUVpeak and coefficient of variation (CoV) values of 18F-Fluciclovine uptake are consistent with tumour heterogeneity revealed by immunohistochemistry. We studied the expression of amino acid transporters (AATs) for 18F-Fluciclovine, namely LAT1 (SLC7A5 and SLC3A2) and ASCT2 (SLC1A5). SLC7A5 and SLC3A2 were expressed at relatively high levels in 22Rv1 castration-resistant orthografts following chronic ADT (modelling clinical castration-resistant disease), while SLC1A5 was preferentially expression in CWR22Res tumours following acute ADT. Additional AATs such as SLC43A2 (LAT4) were shown to be upregulated following chronic ADT by transcriptomic analysis; their role in Fluciclovine uptake warrants investigation. Conclusion We studied in vivo 18F-Fluciclovine uptake in human prostate cancer orthograft models following acute and chronic ADT. 18F-Fluciclovine uptakes highlight tumour heterogeneity that may explain castration resistance and can be exploited as a clinical biomarker.

The thieno[2,3-d]pyrimidinedione core is found as a component in a range of pharmaceutically active compounds, however, synthetic approaches to these scaffolds rely on access to functionalised, highly substituted thiophenes. Here we describe a new approach for the preparation of 5-carboxamide-6-aryl analogues that involves a two-step synthesis of the thieno[2,3-d]pyrimidinedione core from a readily available mercaptouracil derivative. Thio-alkylation with ethyl 3-bromopyruvate, followed by cyclisation and dehydration mediated by polyphosphoric acid allowed the scalable synthesis of the thieno[2,3-d] pyrimidinedione unit. The late-stage functionalisation of this core motif via bromination of the thiophene ring and a subsequent Suzuki-Miyaura reaction as the key steps permitted access to a novel library of 5-carboxamide-6-aryl analogues. The physicochemical properties of these compounds were determined, generating an insight into the potential bioavailability of these scaffolds. Based on these results, a selection of the novel 5-carboxamide-6-aryl analogues were tested as lactate uptake inhibitors of monocarboxylate transporters 1, 2 and 4 in Xenopus oocytes.

Poly(ADP-ribose) polymerase (PARP) is involved in repair of DNA breaks and is over-expressed in a wide variety of tumors, making PARP an attractive biomarker for positron emission tomography (PET) and single photon emission computed tomography imaging. Consequently, over the past decade, there has been a drive to develop nuclear imaging agents targeting PARP. Here, we report the discovery of a PET tracer that is based on the potent PARP inhibitor olaparib (1). Our lead PET tracer candidate, [18F]20, was synthesized and evaluated as a potential PARP PET radiotracer in mice bearing subcutaneous glioblastoma xenografts using ex vivo biodistribution and PET–magnetic resonance imaging techniques. Results showed that [18F]20 could be produced in a good radioactivity yield and exhibited specific PARP binding allowing visualization of tumors over-expressing PARP. [18F]20 is therefore a potential candidate radiotracer for in vivo PARP PET imaging.

Radiopharmaceuticals that incorporate radioactive iodine in combination with single‐photon emission computed tomography imaging play a key role in nuclear medicine, with applications in drug development and disease diagnosis. Despite this importance, there are relatively few general methods for the incorporation of radioiodine into small molecules. This work reports a rapid air‐ and moisture‐stable ipso‐iododeboronation procedure that uses NIS in the non‐toxic, green solvent dimethyl carbonate. The fast reaction and mild conditions of the gold‐catalysed method led to the development of a highly efficient process for the radiolabelling of arenes, which constitutes the first example of an application of homogenous gold catalysis to selective radiosynthesis. This was exemplified by the efficient synthesis of radiolabelled meta‐[125I]iodobenzylguanidine, a radiopharmaceutical that is used for the imaging and therapy of human norepinephrine transporter‐expressing tumours.

A simple and effective one-pot tandem procedure that generates aryl iodides from readily available aryl amines via stable diazonium salts has been developed. The operationally simple procedure and mild conditions allow late-stage iodination of a wide range of aryl compounds bearing various functional groups and substitution patterns. A novel synthetic strategy involving the preparation of nitroaryl compounds followed by a chemoselective tin(II) dichloride reduction and the use of the one-pot diazotisation–iodination transformation was also developed. The general applicability of this approach was demonstrated with the preparation of a number of medicinally important compounds including CNS1261, a SPECT imaging agent of the N-methyl-D-aspartate (NMDA) receptor and IBOX, a compound used to detect amyloid plaques in the brain.

An operationally simple, one-pot, two-step tandem procedure that allows the incorporation of radioactive iodine into aryl amines via stable diazonium salts is described. The mild conditions are tolerant of various functional groups and substitution patterns, allowing late-stage, rapid access to a wide range of 125I-labelled aryl compounds and SPECT radiotracers.

Introduction: \ud Positron Emission Tomography (PET) imaging with selective 18 kDa translocator protein (TSPO) radiotracers has contributed to our understanding on the role of inflammation in disease development and progression. With an increasing number of rodent models of human disease and expansion of the preclinical PET imaging base worldwide, accurate quantification of longitudinal rodent TSPO PET datasets is necessary. This is particularly relevant as TSPO PET quantification relies on invasive blood sampling due to lack of a suitable tissue reference region. Here we investigate the kinetics and quantification bias of a novel TSPO radiotracer [18F]AB5186 in rats using automatic, manual and image derived input functions.\ud \ud Methods: \ud [18F]AB5186 was administered intravenously and dynamic PET imaging was acquired over 2 hours. Arterial blood was collected manually to derive a population based input function or using an automatic blood sampler to derive a plasma input function. Manually sampled blood was also used to analyze the [18F]AB5186 radiometabolite profile in plasma and applied to all groups as a population based dataset. Kinetic models were used to estimate distribution volumes (VT) and [18F]AB5186 outcome measure bias was determined.\ud \ud Results: \ud [18F]AB5186 distribution in rats was consistent with TSPO expression and at 2 h post-injection 50% of parent compound was still present in plasma. Population based manual sampling methods and image derived input function (IDIF) underestimated VT by ~50% and 88% compared with automatic blood sampling, respectively. The VT variability was lower when using IDIF versus arterial blood sampling methods and analysis of the Bland-Altman plots showed a good agreement between methods of analysis.\ud \ud Conclusion: \ud Quantification of TSPO PET rodent data using image-derived methods, which are more amenable for longitudinal scanning of small animals, yields outcome measures with reduced variability and good agreement, albeit biased, compared with invasive blood sampling methods.

Poly(ADP-ribose) polymerase (PARP) is involved in repair of DNA breaks and is over-expressed in a wide variety of tumors, making PARP an attractive biomarker for positron emission tomography (PET) and single photon emission computed tomography imaging. Consequently, over the past decade, there has been a drive to develop nuclear imaging agents targeting PARP. Here, we report the discovery of a PET tracer that is based on the potent PARP inhibitor olaparib (1). Our lead PET tracer candidate, [

The translocator protein (TSPO) is an important target for imaging focal neuroinflammation in diseases such as brain cancer, stroke and neurodegeneration, but current tracers for non-invasive imaging of TSPO have important limitations. We present the synthesis and evaluation of a novel 3-fluoromethylquinoline-2-carboxamide, AB5186, which was prepared in eight steps using a one-pot two component indium(III)-catalysed reaction for the rapid and efficient assembly of the 4-phenylquinoline core. Biological assessment and the implementation of a physicochemical study showed AB5186 to have low nanomolar affinity for TSPO, as well as optimal plasma protein binding and membrane permeability properties. Generation of [18F]-AB5186 through 18F incorporation was achieved in good radiochemical yield and subsequent in vitro and ex vivo autoradiography revealed the ability of this compound to bind with specificity to TSPO in mouse glioblastoma xenografts. Initial positron emission tomography imaging of a glioma bearing mouse and a healthy baboon support the potential for [18F]-AB5186 use as a radiotracer for non-invasive TSPO imaging in vivo.

An operationally simple, one-pot, two-step tandem procedure that allows the incorporation of radioactive iodine into aryl amines via stable diazonium salts is described. The mild conditions are tolerant of various functional groups and substitution patterns, allowing late-stage, rapid access to a wide range of

Objective: Pancreatic cancer is a leading cause of cancer-related death in the Western world. Current chemotherapy regimens have modest survival benefit. Thus, novel, effective therapies are required for treatment of this disease.\ud \ud Design: Activating KRAS mutation almost always drives pancreatic tumour initiation, however, deregulation of other potentially druggable pathways promotes tumour progression. PTEN loss leads to acceleration of KrasG12D-driven pancreatic ductal adenocarcinoma (PDAC) in mice and these tumours have high levels of mammalian target of rapamycin (mTOR) signalling. To test whether these KRAS PTEN pancreatic tumours show mTOR dependence, we compared response to mTOR inhibition in this model, to the response in another established model of pancreatic cancer, KRAS P53. We also assessed whether there was a subset of pancreatic cancer patients who may respond to mTOR inhibition.\ud \ud Results: We found that tumours in KRAS PTEN mice exhibit a remarkable dependence on mTOR signalling. In these tumours, mTOR inhibition leads to proliferative arrest and even tumour regression. Further, we could measure response using clinically applicable positron emission tomography imaging. Importantly, pancreatic tumours driven by activated KRAS and mutant p53 did not respond to treatment. In human tumours, approximately 20% of cases demonstrated low PTEN expression and a gene expression signature that overlaps with murine KRAS PTEN tumours.\ud \ud Conclusions: KRAS PTEN tumours are uniquely responsive to mTOR inhibition. Targeted anti-mTOR therapies may offer clinical benefit in subsets of human PDAC selected based on genotype, that are dependent on mTOR signalling. Thus, the genetic signatures of human tumours could be used to direct pancreatic cancer treatment in the future.

Background Although 70% of postmenopausal women suffer from hot flashes the pathophysiology is poorly understood. The serotonin and noradrenaline reuptake inhibitor (SNRI) venlafaxine provides relief of flushing although the mechanism is unknown and could involve a central effect and/or a peripheral effect. Using single photon emission computed tomography (SPECT) we studied the central serotonin transporter (SERT) in vivo using [ 123 I]-beta-carbomethoxy-3-β-(4-iodophenyl)tropane (beta-CIT) and, as previous studies have shown that reactivity of the skin blood vessels is enhanced in those who flush, we examined cutaneous microvascular perfusion. Methods Cutaneous microvascular perfusion was assessed in 31 postmenopausal women, with flushing, using laser Doppler imaging with iontophoresis (LDI + ION), before and after 8 weeks of treatment with venlafaxine. A sub-group of 14 of these women also had SPECT imaging at both time points to evaluate the availability of SERT in the brain. Flush frequency and score was recorded, and Beck Depression Inventory (BDI) II scores were assessed before and after treatment. Results Following treatment with venlafaxine, there was a significant reduction in the [ 123 I]-beta-CIT binding ratio, BDI scores, flushing and endothelial dependent perfusion response. [ 123 I]-Beta-CIT reduction was associated with BDI reduction ( r 2 = 0.54; F = 8.8; p = 0.004), but not flushing reduction or perfusion reduction. Conclusions Venlafaxine resulted in a decrease in BDI II scores with an associated reduction in [ 123 I]-beta-CIT binding in a group of non-depressed women. It also improved flush frequency and severity which may be as a result of decreases seen in enhanced cutaneous microvascular perfusion.

SummaryPrader–Willi syndrome (PWS) is a rare condition because of the deletion of paternal chromosomal material (del PWS), or a maternal uniparental disomy (mUPD PWS), at 15q11-13. Affective psychosis is more prevalent in mUPD PWS. We investigated the relationship between the two PWS genetic variants and brain-stem serotonin transporter (5-HTT) availability in adult humans. Mean brain-stem 5-HTT availability determined by [123I]-beta-CIT single photon emission tomography was lower in eight adults with mUPD PWS compared with nine adults with del PWS (mean difference −0.93, t = −2.85, P = 0.014). Our findings confirm an association between PWS genotype and brain-stem 5-HTT availability, implicating a maternally expressed/paternally imprinted gene, that is likely to account for the difference in psychiatric phenotypes between the PWS variants.Declaration of interestNone.

Rapid and efficient radioiodination of aryl and heteroaryl bromides has been achieved using a nickel(0)-mediated halogen-exchange reaction. This transformation gives direct access to [(123)I]- and [(125)I]-imaging agents for single photon emission computed tomography (SPECT), such as 5-[(123)I]-A85380 (see scheme, Boc = tert-butyloxycarbonyl, cod = 1,5-cyclooctadiene, TFA = trifluoroacetic acid).

A focused library of novel 2,3-dihydro-1H-1,5-benzodiazepin-2-ones containing sites for 11C-, 18F- and 123I-labelling have been prepared and evaluated against membrane expressing human recombinant metabotropic glutamate 2 receptor (mGluR2). The compounds were found to be non-competitive antagonists with nanomolar affinity. HPLC evaluation of the physiochemical properties of these compounds identified two candidates for PET and SPECT imaging of mGluR2.

We demonstrate the use of the miniaturised Medipix positron sensor for detection of the clinical PET radiotracer, [(68)Ga]gallium-citrate, on a silica-based monolith, towards microfluidic quality control. The system achieved a far superior signal-to-noise ratio compared to conventional sodium iodide-based radio-HPLC detection and allowed real-time visualisation of positrons in the monolith.

A mild and rapid method for the iodination of arenes that utilizes silver(I) triflimide as a catalyst for activation of N-iodosuccinimide has been developed. The transformation was found to be general for a wide range of anisole, aniline, acetanilide, and phenol derivatives and allowed the late-stage iodination of biologically active compounds such as PIMBA, a SPECT imaging agent of breast cancer, and (−)-IBZM, a dopamine D2 receptor antagonist. The method was also modified for the radioiodination of arenes using a one-pot procedure involving the in situ generation of [125I]-N-iodosuccinimide followed by the silver(I)-catalyzed iodination.

A mild and rapid method for the iodination of arenes that utilizes silver(I) triflimide as a catalyst for activation of N-iodosuccinimide has been developed. The transformation was found to be general for a wide range of anisole, aniline, acetanilide, and phenol derivatives and allowed the late-stage iodination of biologically active compounds such as PIMBA, a SPECT imaging agent of breast cancer, and (-)-IBZM, a dopamine D2 receptor antagonist. The method was also modified for the radioiodination of arenes using a one-pot procedure involving the in situ generation of [(125)I]-N-iodosuccinimide followed by the silver(I)-catalyzed iodination.

Goals:This paper presents the performance review based on a dual-ring Positron Emission Tomography (PET) scanner being a part of Bruker Albira: a multi-modal small-animal imaging platform. Each ring of Albira PET contains eight detectors arranged as octagon, and each detector is built using a single continuous lutetium-yttrium oxyorthosilicate crystal and multi-anode photo multiplier tube. In two-ring configuration, the scanner covers 94.4 mm in axial- and 80´80 mm in trans-axial direction, which is sufficient to acquire images of small animals (e.g.mice) without the need of moving the animal bed during the scan.Methods:All measurements and majority of data processing were performed according to the NEMA NU4-2008 standard with one exception. Due to the scanner geometry, the spatial resolution test was reconstructed using iterative algorithm instead of the analytical one. The main performance characteristics were compared with those of the other PET sub-systems of tri-modal small-animal scanners.Results:The measured spatial resolution at the centre of the axial field of view in radial, tangential and axial directions was 1.72, 1.70 and 2.45 mm, respectively. The scatter fraction for the mouse-like phantom was 9.8% and for the rat-like phantom, 21.8%. The maximum absolute sensitivity was 5.30%. Finally, the recovery co-efficients for 5, 4, 3, 2, 1 mm diameter rods in image quality phantom were: 0.90, 0.77, 0.66, 0.30 and 0.05, respectively.Conclusion:The Bruker Albira is a versatile small-animal multi-modal device that can be used for variety of studies. Overall the PET sub-system provides a good spatial resolution coupled with better-than average sensitivity and the ability to produce good quality animal images when administering low activities.

An iron(III)-catalyzed method for the rapid and highly regioselective iodination of arenes has been developed. Use of the powerful Lewis acid, iron(III) triflimide, generated in situ from iron(III) chloride and a readily available triflimide-based ionic liquid allowed activation of N-iodosuccinimide (NIS) and efficient iodination under mild conditions of a wide range of substrates including biologically active compounds and molecular imaging agents.

Preclinical studies demonstrate that pro-inflammatory cytokines increase serotonin transporter availability and function, leading to depressive symptoms in rodent models. Herein we investigate associations between circulating inflammatory markers and brainstem serotonin transporter (5-HTT) availability in humans. We hypothesised that higher circulating inflammatory cytokine concentrations, particularly of tumour necrosis factor (TNF-α), would be associated with greater 5-HTT availability, and that TNF-α inhibition with etanercept (sTNFR:Fc) would in turn reduce 5-HTT availability. In 13 neurologically healthy adult women, plasma TNF-α correlated significantly with 5-HTT availability (rho=0.6; p=0.03) determined by [123I] -beta-CIT SPECT scanning. This association was replicated in an independent sample of 12 patients with psoriasis/psoriatic arthritis (rho=0.76; p=0.003). Indirect effects analysis, showed that there was a significant overlap in the variance explained by 5-HTT availability and TNF-α concentrations on BDI scores. Treatment with etanercept for 6-8 weeks was associated with a significant reduction in 5-HTT availability (Z= 2.09; p=0.03; r=0.6) consistent with a functional link. Our findings confirm an association between TNF-α and 5-HTT in both the basal physiological and pathological condition. Modulation of both TNF-α and 5-HTT by etanercept indicate the presence of a mechanistic pathway whereby circulating inflammatory cytokines are related to central nervous system substrates underlying major depression.

Targeted radiotherapy using 131I-metaiodobenzylguanidine (131I-MIBG) has produced remissions in some neuroblastoma patients. We previously reported that combining 131I-MIBG with the topoisomerase I inhibitor topotecan induced long-term DNA damage and supraadditive toxicity to noradrenaline transporter (NAT)–expressing cells and xenografts. This combination treatment is undergoing clinical evaluation. This present study investigated the potential of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP-1) inhibition, in vitro and in vivo, to further enhance 131I-MIBG/topotecan efficacy. Methods: Combinations of topotecan and the PARP-1 inhibitor PJ34 were assessed for synergism in vitro by combination-index analysis in SK-N-BE(2c) (neuroblastoma) and UVW/NAT (NAT-transfected glioma) cells. Three treatment schedules were evaluated: topotecan administered 24 h before, 24 h after, or simultaneously with PJ34. Combinations of PJ34 and 131I-MIBG and of PJ34 and 131I-MIBG/topotecan were also assessed using similar scheduling. In vivo efficacy was measured by growth delay of tumor xenografts. We also assessed DNA damage by γH2A.X assay, cell cycle progression by fluorescence-activated cell sorting analysis, and PARP-1 activity in treated cells. Results: In vitro, only simultaneous administration of topotecan and PJ34 or PJ34 and 131I-MIBG induced supraadditive toxicity in both cell lines. All scheduled combinations of PJ34 and 131I-MIBG/topotecan induced supraadditive toxicity and increased DNA damage in SK-N-BE(2c) cells, but only simultaneous administration induced enhanced efficacy in UVW/NAT cells. The PJ34 and 131I-MIBG/topotecan combination treatment induced G2 arrest in all cell lines, regardless of the schedule of delivery. In vivo, simultaneous administration of PJ34 and 131I-MIBG/topotecan significantly delayed the growth of SK-N-BE(2c) and UVW/NAT xenografts, compared with 131I-MIBG/topotecan therapy. Conclusion: The antitumor efficacy of topotecan, 131I-MIBG, and 131I-MIBG/topotecan combination treatment was increased by PARP-1 inhibition in vitro and in vivo.

Oncolytic herpes viruses show promise for cancer treatment. However, it is unlikely that they will fulfill their therapeutic potential when used as monotherapies. An alternative strategy is to use these viruses not only as oncolytic agents but also as a delivery mechanism of therapeutic transgenes to enhance tumor cell killing. The herpes simplex virus 1 deletion mutant HSV1716 is a conditionally replicating oncolytic virus that selectively replicates in and lyses dividing tumor cells. It has a proven safety profile in clinical trials and has demonstrated efficacy as a gene-delivery vehicle. To enhance its therapeutic potential, we have engineered HSV1716 to convey the noradrenaline transporter (NAT) gene (HSV1716/NAT), whose expression endows infected cells with the capacity to accumulate the noradrenaline analog metaiodobenzylguanidine (MIBG). Thus, the NAT gene–infected cells are susceptible to targeted radiotherapy using radiolabeled 131I-MIBG, a strategy that has already shown promise for combined targeted radiotherapy–gene therapy in cancer cells after plasmid-mediated transfection. Methods: We used HSV1716/NAT as a dual cell lysis–gene delivery vehicle for targeting the NAT transgene to human tumor xenografts in vivo. Results: In tumor xenografts that did not express NAT, intratumoral or intravenous injection of HSV1716/NAT induced the capacity for active uptake of 131I-MIBG. Administration of HSV1716/NAT and 131I-MIBG resulted in decreased tumor growth and enhanced survival relative to injection of either agent alone. Efficacy was dependent on the scheduling of delivery of the 2 agents. Conclusion: These findings support a role for combination radiotherapy–gene therapy for cancer using HSV1716 expressing the NAT transgene and targeted radionuclide therapy.

123I-labelled mZIENT (2β-carbomethoxy-3β-(3′-((Z)-2-iodoethenyl)phenyl)nortropane) has been developed as a radioligand for the serotonin transporter. The aim of this preliminary study was to assess its whole-body biodistribution in humans and estimate dosimetry. Three healthy controls and three patients receiving selective serotonin reuptake inhibitor (SSRI) therapy for depression were included (two men, four women, age range 41–56 years). Whole-body imaging, brain SPECT imaging and blood and urine sampling were performed. Whole-body images were analysed using regions of interest (ROIs), time–activity curves were derived using compartmental analysis and dosimetry estimated using OLINDA software. Brain ROI analysis was performed to obtain specific-to-nonspecific binding ratios in the midbrain, thalamus and striatum. Initial high uptake in the lungs decreased in later images. Lower uptake was seen in the brain, liver and intestines. Excretion was primarily through the urinary system. The effective dose was estimated to be of the order of 0.03 mSv/MBq. The organ receiving the highest absorbed dose was the lower large intestine wall. Uptake in the brain was consistent with the known SERT distribution with higher specific-to-nonspecific binding in the midbrain, thalamus and striatum in healthy controls compared with patients receiving SSRI therapy. 123I-mZIENT may be a promising radioligand for imaging the serotonin transporters in humans with acceptable dosimetry.

The histamine H3 receptor is implicated in many neurological and psychological disorders and is therefore of interest as a target for pharmacological intervention. The drug development process can be facilitated by using a radiotracer for this receptor in conjunction with single-photon emission computed tomography or positron emission tomography imaging studies, such as drug occupancy studies. This study investigates methods for the radiosynthesis of three compounds to be evaluated as novel radiotracers for the histamine H3 receptor. The radiosyntheses of the three target compounds, (123)I-WYE230949, (123)I-WYE126734 and (123)I-WYE127044 were evaluated using electrophilic iododesilylation and iododestannylation of the corresponding trimethylsilyl and tributylstannyl precursors. All three compounds could be produced in high radiochemical yield by electrophilic iododestannylation. By contrast, only two of the three (WYE230949 and WYE126734) could be produced by electrophilic iododesilylation. This is because of the fact that the trimethylsilyl precursors are less reactive than the stannyl precursors. Electrophilic iododestannylation of the tributylstannyl precursors is therefore the method of choice for radiosynthesis of these compounds because all three target compounds could be produced in high radiochemical yield.

Dysregulation of noradrenergic function has been implicated in a variety of psychiatric and neurodegenerative disorders, including depression and Alzheimer's disease. The noradrenaline transporter (NAT) is a major target for antidepressant drugs, including reboxetine, a selective noradrenaline reuptake inhibitor. Therefore, the development of a radiotracer for imaging of the NAT is desirable. In this study, NKJ64, a novel iodinated analog of reboxetine, was radiolabeled and evaluated as a potential single photon emission computerized tomography (SPECT) radiotracer for imaging the NAT in brain. Biological evaluation of the novel radiotracer, ¹²³/¹²⁵I-NKJ64, was carried out in rats using: in vitro ligand binding assays; in vitro and ex vivo autoradiography; in vivo biodistribution studies and ex vivo pharmacological blocking studies. ¹²⁵I-NKJ64 displayed saturable binding with high affinity for NAT in cortical homogenates (K(D) = 4.82 ± 0.87 nM, mean ± SEM, n = 3). In vitro and ex vivo autoradiography showed the regional distribution of ¹²³I-NKJ64 binding to be consistent with the known density of NAT in brain. Following i.v. injection there was rapid uptake of ¹²³I-NKJ64 in brain, with maximum uptake of 2.93% ± 0.14% (mean ± SEM, n = 3) of the injected dose. The specific to nonspecific ratio (locus coeruleus:caudate putamen) of ¹²³I-NKJ64 uptake measured by ex vivo autoradiography was 2.8 at 30 min post i.v. injection. The prior administration of reboxetine significantly reduced the accumulation of ¹²³I-NKJ64 in the locus coeruleus (>50% blocking). The data indicate that further evaluation of ¹²³I-NKJ64 in nonhuman primates is warranted in order to determine its utility as a SPECT radiotracer for imaging of NAT in brain.

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are noninvasive molecular imaging techniques that can be used to provide functional information in the living human body. They use radiolabeled compounds that bind to specific sites on a biological target, known as radiotracers, enabling us to visualize and characterize specific aspects of a biological process. This chapter describes these techniques and how they can be utilized in the drug development process. Keywords: drug development; imaging; PET; SPECT

Purpose: In clinical molecular imaging the interaction between antidepressant medication and SPECT ligands is a significant potential confound. This study measured nAChR availability, as determined by SPECT imaging, on and off selective serotonin reuptake inhibitors in first episode depressed patients. Methods: Five patients in their first episode of major depressive disorder (MDD) on a single SSRI underwent [123I]5-I-A85380- SPECT neuroimaging prior to stopping their medication and again 6 weeks following medication cessation. Autoradiography of post mortem brain tissue with [125I]5-I-A85380 in the presence or absence of four commonly prescribed antidepressants was also assessed. Results: SSRI antidepressants did not affect the relative binding availability of α4β2 nicotinic acetylcholine receptors for the [123I]5-I-A85380 ligand in vivo. Radioligand binding in vitro was unaffected by a single, high pharmacological concentration of antidepressants. Conclusion: SPECT imaging studies using [123I]5-I-A85380 to measure α4β2 nAChR availability in depressed patients are unlikely to be confounded to a major degree by concurrent antidepressant medication. Synapse 64:111–116, 2010. © 2009 Wiley-Liss, Inc.

Summary Purpose: Carbamazepine (CBZ) is a first-line antiepileptic agent with mood-stabilizing effects in bipolar disorder. It has been reported to influence extracellular concentrations of serotonin and dopamine, suggesting an interaction with monoamine transporters. We have investigated this effect using in vivo single photon emission computed tomography (SPECT) in rats. Methods: Adult male rats received 3 mg/kg/h CBZ via mini-osmotic pump. After 14 days continuous treatment, animals underwent two consecutive SPECT scans, using 125I β-CIT as a radiotracer to label serotonin transporter (SERT) and dopamine transporter (DAT) sites in the brain. Pharmacologic distinction was enabled by 125I β-CIT SPECT imaging in rats acutely exposed to the serotonin and dopamine transporter inhibitors, fluoxetine and GBR12909. The interaction between CBZ and 125I β-CIT binding to SERT and DAT was investigated using in vitro autoradiography. Results: Carbamazepine (10 μm) did not affect binding of 125I β-CIT to isolated rat brain slices, thereby excluding a direct effect on ligand binding to SERT and DAT. SPECT studies with fluoxetine and GBR12909 highlighted SERT binding in thalamus, hippocampus, centromedial nuclei, and occipital cortex, and DAT binding in the caudate. Prolonged treatment with CBZ failed to influence 125I β-CIT binding to either SERT or DAT in any of the brain regions examined. Discussion: This study employed the novel technique of small animal SPECT imaging to investigate the effects of CBZ on monoamine transporters in rat brain. Following prolonged treatment, the drug was without effect on SERT or DAT availability. The mechanism by which CBZ exerts its mood stabilizing effects remains elusive.

Introduction To investigate in vivo differences in the distribution of α4β2 subtypes of nAChR using the ligand 123 I-5-Iodo-3-[2( S )-2-azetidinylmethoxy] pyridine (5IA-85380) and single photon emission computed tomography (SPECT) in DLB and similarly aged controls. Methods Thirty-one subjects (15 DLB and 16 controls) underwent 123 I-5IA-85380 and perfusion ( 99m Tc-exametazime) SPECT scanning. Patient scans were compared to scans of control subjects on a voxel-by-voxel basis using SPM2. Results Compared to controls, significant reductions in relative 123 I-5IA-85380 uptake were identified in frontal, striatal, temporal and cingulate regions in DLB. Elevation of scaled 123 I-5IA-85380 uptake in occipital cortex was observed in DLB relative to controls, as well as being associated with DLB subjects with a recent history of visual hallucinations. Changes in 123 I-5IA-85380 SPECT in DLB were different from perfusion. Conclusion Reductions in normalised 123 I-5IA-85380 uptake in DLB were distinct from their perfusion deficits. Significant increase in occipital lobe uptake was present in DLB, a change most pronounced in subjects with a recent history of visual hallucinations. The findings directly link cholinergic changes in occipital lobe to visual hallucinations in DLB.

In order to image nAChR density in vivo in humans, [5- 123 I]-iodo-3-[2(S)-2-azetidinylmethoxy]pyridine ([5- 123 I]A85380] has been developed as a SPECT imaging tracer. This article outlines the radiochemical synthesis, in vitro validation in human post-mortem tissue and some initial in vivo clinical studies using the ligand [5- 123/125 I]A85380. Limitations of the usefulness of the tracer for routine clinical use are also discussed.

Interest in nuclear imaging of poly(ADP-ribose) polymerase-1 (PARP-1) has grown in recent years due to the ability of PARP-1 to act as a biomarker for glioblastoma and increased clinical use of PARP-1 inhibitors. This study reports the identification of a lead iodinated analog 5 of the clinical PARP-1 inhibitor olaparib as a potential single-photon emission computed tomography (SPECT) imaging agent. Compound 5 was shown to be a potent PARP-1 inhibitor in cell-free and cellular assays, and it exhibited mouse plasma stability but approximately 3-fold greater intrinsic clearance when compared to olaparib. An (123)I-labeled version of 5 was generated using solid state halogen exchange methodology. Ex vivo biodistribution studies of [(123)I]-5 in mice bearing subcutaneous glioblastoma xenografts revealed that the tracer had the ability to be retained in tumour tissue and bind to PARP-1 with specificity. These findings support further investigations of [(123)I]-5 as a non-invasive PARP-1 SPECT imaging agent.

An iron(III)-catalyzed method for the rapid and highly regioselective iodination of arenes has been developed. Use of the powerful Lewis acid, iron(III) triflimide, generated in situ from iron(III) chloride and a readily available triflimide-based ionic liquid allowed activation of N-iodosuccinimide (NIS) and efficient iodination under mild conditions of a wide range of substrates including biologically active compounds and molecular imaging agents.

Herpes simplex virus 1716 (HSV1716), a selectively replication competent mutant of HSV1, is under investigation as an oncolytic viral therapy in human malignant glioma. As with similar therapies, a technique for measurement of viral replication and distribution over time following virus administration is required. Imaging expression of the HSV-thymidine kinase (HSV-tk) gene offers an opportunity for non-invasive assessment of viral distribution in living subjects. This is the first study to explore the use of HSV-tk as a reporter gene and radiolabelled thymidine analogue 5-[(123)I]iodo-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) uracil ((123)I-FIAU) as a marker substrate to non-invasively monitor HSV1716 replication in humans during treatment of high-grade glioma.I-FIAU brain SPECT imaging was undertaken in eight patients receiving intra-tumoural injection of HSV1716, before and after administration of the virus. Baseline images were acquired 3 days prior to virus administration and between 1 and 5 days following virus administration. Region of interest analysis was used to investigate whether there was an increase in (123)I-FIAU concentration following virus administration due to HSV-tk expression.Increased (123)I-FIAU accumulation due to HSV-tk expression was not detected in this study. The possible explanations for this finding are explored and design options for future studies are discussed.

Over the last 20 years a number of radiotracers that target various neurotransmitter systems have been developed for use in imaging studies in psychiatry, but there are many more targets still to be investigated. The development of a radiotracer for clinical positron emission tomography (PET) or single photon emission computed tomography (SPECT) neuroimaging studies can be a complex and lengthy process with few imaging agents successfully progressing into clinical human studies. One of the most challenging aspects in the procedure is the development of a rapid and simple radiosynthesis protocol to obtain the potential radiotracer with adequate specific activity, isolated radiochemical yield and radiochemical purity for human imaging. Once a candidate has been radiolabelled, full characterization of the radiotracer is required before it can be used in clinical human studies. Pre-clinical studies include investigation into the binding distribution, pharmacokinetics, metabolism, toxicology and dosimetry of a radiotracer. There are many points during the development procedure where a potential radiotracer can be rejected. Due to interspecies differences the development of a radiotracer can either go too far with an unsuccessful candidate or can potentially lead to rejection of a candidate too soon. It is only when the radiotracer has been used in humans can we be certain that a radiotracer is a useful imaging agent for clinical research studies. The development of new technologies, such as micro-PET or SPECT can only improve our ability to predict the success of a radiotracer.

The effect of age on brain muscarinic receptor density is unclear. Some in vivo neuroimaging studies have reported a large age-related reduction in muscarinic receptor density; however, others have reported increases or no change. The variability in these results most likely arises because of the heterogeneity of the populations studied, differences in quantification methods employed, and a paucity of subtype selective ligands. Thus, we used the m1/m4 selective probe (R,R)[123I]-I-QNB to investigate age-related differences in brain muscarinic receptors in healthy females. We included 10 younger subjects (age range 26–37) and 22 older women (age range 57–82 years). The older women had significantly lower (R,R)[123I]-I-QNB binding in widespread brain regions including cerebral cortex and hippocampus. Across all subjects, regional binding was significantly negatively correlated with age. Thus, in this population of healthy women, there was an age-related reduction in muscarinic receptor density. This may contribute to age-related differences in cognitive function and risk for Alzheimer's disease.

Nicotinic acetylcholine receptors (nAChRs) have been implicated in a number of neurological disorders. 5-Iodo-3-[2(S)-2-azetidinylmethoxy]pyridine (5-I-A-85380) is a novel nAChR marker, binding predominantly to the alpha4beta2 subtype. This in vitro autoradiography study describes the distribution of 5-[(125)I]-A-85380 binding in post-mortem brain tissue from normal elderly individuals and from cases with age-associated dementias of both neurodegenerative and vascular types. The binding distribution of 5-[(125)I]-A-85380 in normal brain tissue was found to be consistent with the reported distribution of other high-affinity nicotinic ligands. In addition to high thalamic and moderate striatal and temporal cortex density, moderate 5-[(125)I]-A-85380 binding was also seen in white matter tracts in cingulate, occipital, and temporal areas, indicating the presence of nAChRs along nerve fiber tracts, which has not been reported in other high-affinity nicotinic agonist distribution studies. In Parkinson's disease (PD), loss of striatal 5-[(125)I]-A-85380 binding closely parallels the loss of nigrostriatal dopaminergic markers previously observed. In dementia with Lewy bodies (DLB) reduced striatal 5-[(125)I]-A-85380 binding density, comparable to that in PD, may be a marker of early degeneration in nigrostriatal inputs, while in Alzheimer's disease (AD) reduced striatal 5-[(125)I]-A-85380 binding could be related to reduced cortical inputs. The reductions of nAChRs seen in AD, DLB, and PD were not apparent in vascular dementia (VaD). In conclusion, 5-I-A-85380 is clearly a useful ligand for both in vitro and in vivo single photon emission tomography human studies investigating disease symptoms and progression, response to acetylcholinesterase-inhibiting drugs and in differentiating primary degenerative dementia from VaD.

OBJECTIVES: To image amyloid deposition in patients with traumatic brain injury (TBI) using carbon 11-labeled Pittsburgh Compound B ([11C]PiB) positron emission tomography (PET) and to validate these findings using tritium-labeled PiB ([3H]PiB) autoradiography and immunocytochemistry in autopsy-acquired tissue. DESIGN, SETTING, AND PARTICIPANTS: In vivo PET at tertiary neuroscience referral center and ex vivo immunocytochemistry of autopsy-acquired brain tissue from a neuropathology archive. [11C]PiB PET was used to image amyloid deposition in 11 controls (median [range] age, 35 [24-60] years) and in 15 patients (median [range] age, 33 [21-50] years) between 1 and 361 days after a TBI. [3H]PiB autoradiography and immunocytochemistry for β-amyloid (Aβ) and β-amyloid precursor protein in brain tissue were obtained from separate cohorts of 16 patients (median [range] age, 46 [21-70] years) who died between 3 hours and 56 days after a TBI and 7 controls (median [range] age, 61 [29-71] years) who died of other causes. MAIN OUTCOMES AND MEASURES: We quantified the [11C]PiB distribution volume ratio and standardized uptake value ratio in PET images. The distribution volume ratio and the standardized uptake value ratio were measured in cortical gray matter, white matter, and multiple cortical and white matter regions of interest, as well as in striatal and thalamic regions of interest. We examined [3H]PiB binding and Aβ and β-amyloid precursor protein immunocytochemistry in autopsy-acquired brain tissue. RESULTS: Compared with the controls, the patients with TBI showed significantly increased [11C]PiB distribution volume ratios in cortical gray matter and the striatum (corrected P < .05 for both), but not in the thalamus or white matter. Increases in [11C]PiB distribution volume ratios in patients with TBI were seen across most cortical subregions, were replicated using comparisons of standardized uptake value ratios, and could not be accounted for by methodological confounders. Autoradiography revealed [3H]PiB binding in neocortical gray matter, in regions where amyloid deposition was demonstrated by immunocytochemistry; white matter showed Aβ and β-amyloid precursor protein by immunocytochemistry, but no [3H]PiB binding. No plaque-associated amyloid immunoreactivity or [3H]PiB binding was seen in cerebellar gray matter in autopsy-acquired tissue from either controls or patients with TBI, although 1 sample of cerebellar tissue from a patient with TBI showed amyloid angiopathy in meningeal vessels. CONCLUSIONS AND RELEVANCE: [11C]PiB shows increased binding following TBI. The specificity of this binding is supported by neocortical [3H]PiB binding in regions of amyloid deposition in the postmortem tissue of patients with TBI. [11C]PiB PET could be valuable in imaging amyloid deposition following TBI.

Disturbances in the cerebral cholinergic nervous system and subsequent reduction of acetylcholine (ACh) are neurochemical features of Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB), and treatments with acetylcholinesterase inhibitors (AChEIs) offer symptomatic improvement in cognitive and non-cognitive symptoms. One important subclass of ACh receptors are nicotinic acetylcholine receptors (nAChRs), which have been shown to be implicated in memory and cognitive processes. The SPECT tracer 123I-5IA-85380 is a radioligand that can visualise in vivo the presynaptic nAChR (α4β2 subtype, the most common subtype in man).1 In the present study, we assessed the effects of the AChEI donepezil on nicotinic receptor status in AD and DLB using 123I-5IA-85380 SPECT imaging. We hypothesised that relative 123I-5IA-85380 uptake would increase after administration of donepezil in accordance with previous studies using non-selective nicotinic ligands.2 ### Subjects We recruited 9 non-smoking patients (for >10 years) with dementia (4 AD and 5 DLB). Patients with AD fulfilled NINCDS/ADRDA criteria for “probable” AD,3 whereas DLB subjects satisfied criteria for “probable” DLB.4 The study received ethical and ARSAC approval. All participants and their nearest relative gave informed written consent. ### Assessments and study design Prior to baseline 123I-5IA-85380 imaging, patients underwent physical, neurological and neuropsychiatric assessments. Tests included the Cambridge Cognitive Examination (CAMCOG), the Mini-Mental State Examination (MMSE) and the motor Unified Parkinson’s Disease Rating Scale (UPDRS III). Following …

A new synthesis of the peripheral benzodiazepine receptor ligand, PK11195 has been developed in only six-steps using a Heck-type reaction and a Suzuki coupling to effect the key transformations. The flexibility of this new approach is demonstrated by the synthesis of an iodo-analogue of PK11195 prepared from the corresponding bromide using a copper catalysed aromatic Finkelstein reaction.

Although 70% of postmenopausal women suffer from hot flashes the pathophysiology is poorly understood. The serotonin and noradrenaline reuptake inhibitor (SNRI) venlafaxine provides relief of flushing although the mechanism is unknown and could involve a central effect and/or a peripheral effect. Using single photon emission computed tomography (SPECT) we studied the central serotonin transporter (SERT) in vivo using [(123)I]-beta-carbomethoxy-3-β-(4-iodophenyl)tropane (beta-CIT) and, as previous studies have shown that reactivity of the skin blood vessels is enhanced in those who flush, we examined cutaneous microvascular perfusion.Cutaneous microvascular perfusion was assessed in 31 postmenopausal women, with flushing, using laser Doppler imaging with iontophoresis (LDI+ION), before and after 8 weeks of treatment with venlafaxine. A sub-group of 14 of these women also had SPECT imaging at both time points to evaluate the availability of SERT in the brain. Flush frequency and score was recorded, and Beck Depression Inventory (BDI) II scores were assessed before and after treatment.Following treatment with venlafaxine, there was a significant reduction in the [(123)I]-beta-CIT binding ratio, BDI scores, flushing and endothelial dependent perfusion response. [(123)I]-Beta-CIT reduction was associated with BDI reduction (r(2)=0.54; F=8.8; p=0.004), but not flushing reduction or perfusion reduction.Venlafaxine resulted in a decrease in BDI II scores with an associated reduction in [(123)I]-beta-CIT binding in a group of non-depressed women. It also improved flush frequency and severity which may be as a result of decreases seen in enhanced cutaneous microvascular perfusion.

Preliminary investigation of the radioiodinated (S,S)-reboxetine analogue, 123I-INER, in baboons showed this tracer to have promise for imaging the noradrenaline transporter (NAT) using single photon emission computed tomography (SPECT). More recently, the radioiodinated (R,S)-stereoisomer of 123I-INER, 123I-NKJ64, has been synthesized and preliminary evaluation in rats has been reported. This article reports the brain distribution and pharmacokinetic properties of 123I-NKJ64 in baboons and compares results with 123I-INER data in the same species. SPECT studies were conducted in two ovariectomized adult female baboons using two different protocols: (1) bolus of 123I-INER or 123I-NKJ64; and (2) bolus plus constant infusion of 123I-NKJ64 with reboxetine (2.0 mg/kg) administration at equilibrium. Following bolus injection, both radiotracers rapidly and avidly entered the baboon brain. The regional brain accumulation of 123I-NKJ64 did not match the known distribution of NAT in baboon brain, contrasting with previous results obtained in rats. Conversely, the regional distribution of 123I-INER was consistent with known distribution of NAT in baboon brain. No displacement of 123I-NKJ64 was observed following administration of reboxetine. This contrasts with previous data obtained for 123I-INER, where 60% of specific binding was displaced by a lower dose of reboxetine. These data suggest that 123I-NKJ64 may lack affinity and selectivity for NAT in baboon brain and 123I-INER is the most promising iodinated reboxetine analogue developed to date for in vivo imaging of NAT in brain using SPECT. This study highlights the importance of species differences during radiotracer development and the stereochemical configuration of analogues of reboxetine in vivo. Synapse 66:923–930, 2012. © 2012 Wiley Periodicals, Inc.