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Parkinson’s disease (PD) is a neurodegenerative condition with several major hallmarks, including loss ofsubstantia nigraneurons, reduction in striatal dopaminergic function, and formation of α-synuclein-rich Lewy bodies. Mutations inSNCA, encoding for α-synuclein, are a known cause of familial PD, and the G51D mutation causes a particularly aggressive form of the condition. CRISPR/Cas9 technology was used to introduce the G51D mutation into the endogenous ratSNCAgene.SNCAG51D/+andSNCAG51D/G51Drats were born in Mendelian ratios and did not exhibit any severe behavourial defects.L-3,4-dihydroxy-6-18F-fluorophenylalanine (18F-DOPA) positron emission tomography (PET) imaging was used to investigate this novel rat model. Wild-type (WT),SNCAG51D/+andSNCAG51D/G51Drats were characterised over the course of ageing (5, 11, and 16 months old) using18F-DOPA PET imaging and kinetic modelling. We measured the influx rate constant (Ki) and effective distribution volume ratio (EDVR) of18F-DOPA in the striatum relative to the cerebellum in WT,SNCAG51D/+andSNCAG51D/G51Drats. A significant reduction inEDVRwas observed inSNCAG51D/G51Drats at 16 months of age indicative of increased dopamine turnover. Furthermore, we observed a significant asymmetry in EDVR between the left and right striatum in agedSNCAG51D/G51Drats. The increased and asymmetric dopamine turnover observed in the striatum of agedSNCAG51D/G51Drats is similar to prodromal PD, which suggests the presence of compensatory mechanisms.SNCAG51Drats represent a novel genetic model of PD, and kinetic modelling of18F-DOPA PET data has identified a highly relevant early disease phenotype.

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.

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae. Identification of neutrophil activation in real time in the lungs of patients may permit biological stratification of patients in otherwise heterogenous cohorts typically defined by clinical criteria. No methods for identifying neutrophil activation in real time in the lungs of patients currently exist. We developed a bespoke molecular imaging probe targeting three characteristic signatures of neutrophil activation: pinocytosis, phagosomal alkalinisation, and human neutrophil elastase (HNE) activity. The probe functioned as designed in vitro and ex vivo. We evaluated optical endomicroscopy imaging of neutrophil activity using the probe in real-time at the bedside of healthy volunteers, patients with bronchiectasis, and critically unwell mechanically ventilated patients. We detected a range of imaging responses in vivo reflecting heterogeneity of condition and severity. We corroborated optical signal was due to probe function and neutrophil activation.

Dosimetry models using preclinical positron emission tomography (PET) data are commonly employed to predict the clinical radiological safety of novel radiotracers. However, unbiased clinical safety profiling remains difficult during the translational exercise from preclinical research to first-in-human studies for novel PET radiotracers. In this study, we assessed PET dosimetry data of six 18F-labelled radiotracers using preclinical dosimetry models, different reconstruction methods and quantified the biases of these predictions relative to measured clinical doses to ease translation of new PET radiotracers to first-in-human studies. Whole-body PET images were taken from rats over 240 min after intravenous radiotracer bolus injection. Four existing and two novel PET radiotracers were investigated: [18F]FDG, [18F]AlF-NOTA-RGDfK, [18F]AlF-NOTA-octreotide ([18F]AlF-NOTA-OC), [18F]AlF-NOTA-NOC, [18F]ENC2015 and [18F]ENC2018. Filtered-back projection (FBP) and iterative methods were used for reconstruction of PET data. Predicted and true clinical absorbed doses for [18F]FDG and [18F]AlF-NOTA-OC were then used to quantify bias of preclinical model predictions versus clinical measurements. Our results show that most dosimetry models were biased in their predicted clinical dosimetry compared to empirical values. Therefore, normalization of rat:human organ sizes and correction for reconstruction method biases are required to achieve higher precision of dosimetry estimates.

Background Fibrosis is a fundamental process involved in healing and remodelling post myocardial infarction (MI) and during heart failure (HF). Positron emission tomography (PET) is a potential tool for investigating active collagen biosynthesis in the myocardium with fluoroprolines. We hypothesised that 18F-fluoroprolines can identify either triple helix collagen (trans-4-18F-fluoro-L-proline, e.g.scar tissue) or single-chain polypeptide abnormal collagen (cis 4 18F fluoro L proline, e.g.interstitial fibrosis) by directly targeting collagen biosynthesis. Methods Adult male Sprague-Dawley rats underwent total left coronary artery occlusion to induce MI. Seven days after MI, animals underwent magnetic resonance imaging (MRI) and were injected with trans- or cis-4-18F-fluoro-L-prolines for PET imaging. Hearts collected after in vivo imaging were stained with Picrosirius red (PSR) to assess myocardial fibrosis. Another set of rats underwent 4-week angiotensin-II (AngII) treatment (250 ng/kg/min or 500 ng/kg/min), to induce left ventricular hypertrophy and diffuse fibrosis, with blood pressure checks and ex vivo assessment of myocardial collagen content (PSR stain; hydroxyproline (HP) assay), to develop a model of interstitial fibrosis for future PET imaging studies. Results Uptake of trans-fluoroproline was strongest in the infarct area and cis-fluoroproline was strongest in the remote myocardium (mean standard uptake value ratio of 1.3 vs 0.7). The trans-fluoroproline SUVr strongly, and inversely correlated with ventricular ejection fraction (r2=0.93) and scar tissue size, as determined by MRI, and areas of fibrosis determined by PSR staining. Following AngII treatment, blood pressure significantly increased in both treatment groups by week 2 and stayed high until the end of the study in the high dose group compared to baseline. PSR analysis of heart tissue (AngII study-arm) showed a trend towards increased% fibrosis in the myocardium in the high dose group compared to controls. HP levels were significantly increased with high dose treatment compared to controls (mean: 0.7 vs 0.37 µg/mg; ANOVA p Conclusions Fluoroproline PET imaging could identify and quantify fibrosis activity in vivo following MI and the results validated with histological markers of fibrosis. Based on the results so far, the development of the diffuse fibrosis model to mimic hypertensive HF is promising. Future PET imaging studies with 18F-fluoroprolines and the model of hypertensive heart failure are ongoing.

Aims Cardiovascular thrombosis is responsible a quarter of deaths annually worldwide. Current imaging methods for cardiovascular thrombosis focus on anatomical identification of thrombus but cannot determine thrombus age or activity. Molecular imaging techniques hold promise for identification and quantification of thrombosis in vivo. Our objective was to assess a novel optical and positron-emitting probe targeting Factor XIIIa (ENC2015) as biomarker of active thrombus formation. Methods and results Optical and positron-emitting ENC2015 probes were assessed ex vivo using blood drawn from human volunteers and passed through perfusion chambers containing denuded porcine aorta as a model of arterial injury. Specificity of ENC2015 was established with co-infusion of a factor XIIIa inhibitor. In vivo18F-ENC2015 biodistribution, kinetics, radiometabolism, and thrombus binding were characterized in rats. Both Cy5 and fluorine-18 labelled ENC2015 rapidly and specifically bound to thrombi. Thrombus uptake was inhibited by a factor XIIIa inhibitor. 18F-ENC2015 remained unmetabolized over 8 h when incubated in ex vivo human blood. In vivo, 42% of parent radiotracer remained in blood 60 min post-administration. Biodistribution studies demonstrated rapid clearance from tissues with elimination via the urinary system. In vivo,18F-ENC2015 uptake was markedly increased in the thrombosed carotid artery compared to the contralateral patent artery (mean standard uptake value ratio of 2.40 vs. 0.74, P Conclusion ENC2015 rapidly and selectively binds to acute thrombus in both an ex vivo human translational model and an in vivo rodent model of arterial thrombosis. This probe holds promise for the non-invasive identification of thrombus formation in cardiovascular disease.

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.

In Positron Emission Tomography (PET) research, it is important to assess not only pharmacokinetics of a radiotracer in vivo, but also of the drugs used in blocking/displacement PET studies. Typically, pharmacokinetic/pharmacodynamic (PK/PD) analyses of drugs used in rodent PET studies are based on population average pharmacokinetic profiles of the drugs due to limited blood volume withdrawal while simultaneously maintaining physiological homeostasis. This likely results in bias of PET data quantification, including unknown bias of target occupancy (TO) measurements. This study aimed to develop a High Performance Liquid Chromatography (HPLC) method for PK/PD quantification of drugs used in preclinical rodent PET research, specifically the translocator 18 kDa protein (TSPO) selective drug, PK11195, that used sub-millilitre blood volumes. The lowest detection limit for the proposed HPLC method ranged between 7.5 and 10 ng/mL depending on the method used to calculate the limit of detection, and the measured average relative standard deviation for intermediate precision was equal to 17.2%. Most importantly, we were able to demonstrate a significant difference between calculated PK11195 concentrations at 0.5, 1, 2, 3, 5, 15 and 30 min post-administration and individually measured whole blood levels (significance level range from p

Background Identification of in vivo thrombus formation has the potential to assist in the diagnosis and management of acute coronary atherothrombosis. ENC2015 is a 13-amino acid fluorescent peptide analogue of 2-antiplasmin which binds to newly formed thrombus. Our aim was to assess the binding of both optical (Cy5) and PET (18F) labelled ENC2015 acute human thrombus formation in a translational model of deep arterial injury. Methods In healthy volunteers, blood was drawn through 3 ex-vivo perfusion chambers maintained at 37°C. Each chamber contained a denuded strip of porcine aorta as a model of deep arterial injury. In the optical study each subject participated in three sequential runs; Sequence 1 – ENC2015 +blood; Sequence 2- ENC2015 +inhibitor (iodoacetamide) +blood; Sequence 3 – blood only. In the PET study, each participant participated in only sequence 1 and 2. All sequences can be seen in figure 1. Optical Results Macroscopic fluorescence of high shear thrombi in sequence 1 was significantly greater than sequence 2 (C.I −25.47 to −3.45, p=0.016; figure 2, graph C) and 3 (C.I −28.9 to 4.41, p=0.018; figure 2, graph C). Microscopic fluorescence of sequence 1 was greater than sequence 2 in both high shear thrombi (C.I −1862 to −1358, p=0.0001; figure 2, graph A) and low shear thrombi (C.I −4782 to −3236, p=0.0001; figure 2, graph B). Microscopic fluorescence in sequence 1 was greater than 3 in both high shear (CI −2138 to −1638, p=0.0001; figure 2, graph A) and low shear thrombi (C.I −5008 to −3398, p=0.0001; figure 2, graph B). Fluorescence of sequence 1 was also greater than ENC2015 without blood (no thrombus) as well as a factor XIII control agent and a free Cy5 unbound to ENC2015. PET Results Thrombus uptake (KBq/cc) of 18F-ENC2015 was on average 16.93 times greater in low shear thrombi in sequence 1 compared to sequence two. In the first group of high shear thrombi 18F-ENC2015 uptake was on average 9.27 times greater in sequence 1 than sequence 2. Similarly, group 2 high shear thrombi uptake in sequence 1 was on average 5 times greater than sequence 2 (figure 2, graph D). Conclusion Providing real-time characterisation of in vivo human coronary thrombus formation is vital for our appreciation of the pathophysiology of unstable coronary plaque events. We have demonstrated that our optical and PET probe rapidly binds to acutely formed human thrombus in an environment simulated to that of diseased human coronary arteries.

Identification of in vivo thrombus formation has the potential to assist in the diagnosis and management of acute coronary atherothrombosis. ENC2015 is a 13-amino acid peptide Factor XIIIa ligand which binds to newly formed thrombus. Our aim was to assess the binding of both fluorescent and

Background Acute respiratory distress syndrome is a severe and heterogeneous condition. Clinical diagnostic criteria lack specificity and fail to differentiate the various inflammatory phenotypes. Diagnosis and stratification can be improved by profiling the neutrophil and one of its enzymes, neutrophil elastase (NE), within the alveolar space. We aimed to combine fibre-based optical endomicroscopy (OEM) with a bespoke imaging probe to deliver a new strategy to elucidate pathobiological processes in the lung. Methods We designed and synthesised an optical imaging probe termed neutrophil activation probe to fluoresce as it enters the alkaline neutrophilic vacuole and through NE-specific cleavage. Validation was performed with spectrophotometry, confocal microscopy, and flow cytometry. The probe was tested in a perfused large sheep lung model in conjunction with bronchoscopic OEM. Neutrophil activation probe was synthesised to GMP, and a phase 1 study is underway in healthy volunteers and patients in the intensive care unit. The phase 1 study is registered with EudraCT, number 2011-066167-17, and with ClinicalTrials.gov, number NCT01532024. Findings Neutrophil activation probe was neutrophil specific, specific to NE among other serine proteinases, and demonstrated dequenching in alkaline pH. Intracellular fluorescence signal determined by flow cytometry increased in chemically activated neutrophils (mean fluorescence index signal increased from 24 144 (SE 6175) to 1·218 × 10 6 (59 325), p Interpretation We have demonstrated the potential utility of this bedside molecular imaging strategy, from bench to patient: the probe functions in a predictable manner, in keeping with its design. A two-site international phase 2 clinical study is planned, which will assess the test characteristics of this novel technique and its ability to predict and stratify clinically important outcomes in a heterogeneous ventilated population in the intensive care unit. Funding Medical Research Council.