The sphingosine-1-phosphate-5 (S1P5) receptor is one of the five membrane G protein-coupled receptors that are activated by the lysophospholipid, sphingosine-1-phosphate, resulting in regulation of many cellular processes. S1P5 receptors are located on oligodendrocytes and are proposed to influence oligodendrocyte physiology. Understanding S1P5 modulation during processes such as remyelination could have potential applications for demyelinating CNS disorders such as multiple sclerosis (MS). Herein, we report the synthesis and preliminary evaluation of a series of fluorinated 6-arylaminobenzamides as positron emission tomography (PET) ligands of S1P5. Pharmacokinetic screening and binding evaluation using a [35S]GTPγS assay led to the discovery of TEFM78, a selective and high affinity agonist of S1P5. Radiosynthesis of [18F]TEFM78 allowed pilot PET imaging studies in an animal model, which showed that [18F]TEFM78 can cross the blood brain barrier with good uptake in rat brain and spinal cord.

Sphingosine-1-phosphate-5 receptors (S1P(5)) are predominantly expressed in oligodendrocytes and as a result have been proposed as an important target in Multiple Sclerosis (MS). Selective S1P(5) radiotracers could enable vivo positron emission tomography (PET) imaging of oligodendrocytes activity. Here we report the synthesis, radiolabelling and first preclinical evaluation of the pharmacokinetics and binding properties of a lead 6-arylaminobenzamide derivative, 6-(mesitylamino)-2-methoxy-3-methylbenzamide (also named as TEFM180), as potential core scaffold for development of novel S1P(5) PET radiotracers. Following intravenous bolus injection, TEFM180 was found to quickly enter the brain with good brain:blood ratios and subsequent rapid clearance. Autoradiography studies showed that [H-3]TEFM180 had a high affinity for its target (K-D = 2.8 nM), with moderate levels of non-displaceable binding. Distribution of [H-3]TEFM180 in the brain was found to be consistent with S1P(5) expression and showed a binding potential (BP) of >2-3 in white matter rich regions. Overall, TEFM180 offers a good initial platform for development of future radiotracers targeting S1P(5).

The preclinical surgically-induced models of myocardial infarction (MI) have been extensively utilised for studying the wound healing response in the mammalian heart. It is well-established that these invasive procedures induce marked acute systemic inflammation and adverse haemodynamic derangements, which can result in extra-cardiac tissue injury and remodelling; however, the extent of this has not yet been robustly characterised. Here, using a rat permanent coronary artery ligation (PL) model of MI and the cis- and trans-4-[18F]fluoro-l-proline positron emission tomography (PET) probes, targeting active misfolded and triple-helical collagen biosynthesis, respectively, we conducted an exploratory time-course analysis of the associated extra-cardiac tissue remodelling post-injury. Our results showed an increased cis-4-[18F]fluoro-l-proline PET signal in the liver (P = 0.02; two sample t-test) and lung (P = 0.03; two sample t-test) at the acute day 7 timepoint in the MI group compared to the Sham group (n = 3-6), which was not apparent at the later timepoints (days 14, 28, and 84). Ex vivo quantification of the deposited collagen in extra-cardiac tissues using Sirius red histochemical staining and the total hydroxyproline assay validated the apparent time course trends observed with [18F]fluoro-l-proline PET. These findings suggest that the liver and lungs are susceptible to acute injury in the rat PL model of MI and align with numerous clinical reports of extracardiac injury in MI patients. Moreover, our results highlight the suitability of [18F]fluoro-l-proline PET as a systems-based imaging technique for investigating active collagen biosynthesis across multiple organs.

Age, a key cardiovascular disease risk factor, leads to collagen accumulation, driving age-dependent cardiac remodelling. Sex differences in cardiac health are noted, with females more resistant to age-related collagen accumulation until menopause. The role of collagen synthesis in this phenomenon remains poorly understood. This study uses in vitro rat cardiac fibroblast culture and preclinical in vivo Positron Emission Tomography (PET) experiments with cis-4-[18F]fluoro-l-proline (cis-[18F]FPro) & trans-4-[18F]fluoro-l-proline (trans-4-[18F]FPro) radiotracers to explore the impact of aging and sex on cardiac collagen synthesis using rats. Cardiac fibroblasts from male and female rodents were cultured and incubated with cis-4-[18F]FPro or trans-4-[18F]FPro and radiotracer binding was determined. In vivo PET imaging was performed on male (n=9–11) and female (n=9–12) Sprague-Dawley rats at 1, 3, 6 & 12 months using both radiotracers. Standardised uptake values were normalized to the blood pool. The cell study identified a significant increase in cis-[18F]FPro uptake in males vs females but no significant difference in trans-4-[18F]FPro uptake in vitro, suggesting that sex did alter unhydroxylated collagen synthesis in vitro. In aged cells, there was a significant increase in both tracers in vitro indicating that age increased collagen synthesis. Contrastingly, trans-4-[18F]FPro uptake declined in the ventricles with advancing age in vivo in both sexes. This implies that ventricular hydroxylated collagen synthesis remains stable until 12 months but decreases thereafter. Limited age-related variations were noted in cis-4-[18F]FPro uptake, most evident up to 6 months, predominantly affecting the atria, with no changes measured in the ventricles due to age or sex. This study represents the first exploration into atrial collagen synthesis in vivo and identified a clear age-related decline in collagen synthesis in the ventricles. In cultured cardiac fibroblasts, age-related effects on collagen synthesis differ from our in vivo findings, indicating a likely promotion of collagen synthesis mechanisms with age in the culture environment. This work was funded by the British Heart Foundation (FS/19/34/34354), KP is currently funded by the British Heart Foundation 1+3 Studentship at the University of Edinburgh (FS/20/34017). VJMR, AAST, TEFM, and MGM were funded by the British Heart Foundation (RG/16/10/32375, FS/19/34/34354). AAST is a recipient of a Wellcome Trust Technology Development Award (221295/Z/20/Z). LMR is funded by the EPSRC (EP/N509668/1). CA-C is supported by the Edinburgh Preclinical Imaging. The authors are grateful to the Little France BVS staff, the Edinburgh Imaging radiochemistry staff, and the Glasgow Radiotracer Production Unit (RPU) staff.

Acoustically active perfluorocarbon nanodroplets, smaller in size than conventional microbubbles, show significant potential for enhancing utility of contrast ultrasound imaging and drug delivery beyond the confines of the vasculature. Effective exploitation of these nanoscale agents requires detailed understanding of transit and retention in vivo. However, dependence on droplet vaporization for echogenicity and ultrasound tracking presents significant limitation to imaging to support full understanding of nanodroplet drainage, transit and retention times throughout the body. We propose multimodal [F-18]FDG-nanodroplets and demonstrate utility for PET imaging in a xenographic model of colorectal cancer. Microbubbles (MicroMarker, Fujifilm) were prepared for use or condensed to create nanodroplets [1]. Suspensions were diluted 1:5 and held at 36 degrees C in a micro capillary flow system (20 mu L/sec) and subject to increasing acoustic pressures (1 MHz, 10 cycles, PRF 10 ms, 0.25 - 2.5 MPa). Acoustic droplet vaporization was detected by passive cavitation (Precision Acoustics) with signals post-processed in MATLAB. For PET imaging, suspensions were reconstituted with 700 mu L [F-18]FDG, incubated for 1 hour, centrifuged (350 g, 2 minutes), supernatant discarded and resuspended in 700 mu L saline and administered via toe injection to right ([F-18]FDG-microbubbles) or left ([F-18]FDG-nanodroplets) foot of WT mouse (n=3) and imaged using PET/CT (Medison nano) over 60 minutes. PET-nanodroplets were administered to both feet of a tumor bearing (right) colorectal cancer xenographic mouse (n=1). MicroMarker nanodroplets (diameter 129.2 +/- 5.1 nm) were successfully formulated through condensation of microbubbles (1100.0 +/- 180.0 nm), and conversion back into microbubbles confirmed at pressures above 1 MPa. [F-18]FDG-labelling enabled PET imaging (A) and investigation of microbubble and nanodroplet clearance from injection site (B), indicating that microbubbles drained quicker than nanodroplets following toe injection. PET imaging of [F-18]FDG-nanodroplets suggests tumor presence retards nanodroplet clearance from toe injection site as compared to healthy contralateral control (0.6 and 1.8 SUVbw/hour respectively).

Neuroinflammation is associated with a number of brain diseases, making it a common feature of cerebral pathology. Among the best-known biomarkers for neuroinflammation in Positron Emission Tomography (PET) research is the 18 kDa translocator protein (TSPO). This study aims to investigate the binding kinetics of a novel TSPO PET radiotracer, [18F]LW223, in mice and specifically assess its volume of non-displaceable binding (VND) in brain as well as investigate the use of simplified analysis approaches for quantification of [18F]LW223 PET data. Adult male mice were injected with [18F]LW223 and varying concentrations of LW223 (0.003-0.55 mg/kg) to estimate VND of [18F]LW223. Dynamic PET imaging with arterial input function studies and radiometabolite studies were conducted. Simplified quantification methods, standard uptake values (SUV) and apparent volume of distribution (VTapp), were investigated. [18F]LW223 had low VND in the brain (<10% of total binding) and low radiometabolism (∼15-20%). The 2-tissue compartment model provided the best fit for [18F]LW223 PET data, although its correlation with SUV90-120min or VTapp allowed for [18F]LW223 brain PET data quantification in healthy animals while using simpler experimental and analytical approaches. [18F]LW223 has the required properties to become a successful TSPO PET radiotracer.

Inflammation is a key process influencing left ventricular remodelling following myocardial infarction (MI) and can be imaged using Positron Emission Tomography (PET) targeting the 18Da translocator protein (TSPO). We have developed the TSPO radiotracer [18F]LW223, that overcomes several barriers holding back wide adoption of TSPO imaging. This study utilised a rat MI-model to assess whether [18F]LW223 could accurately detect inflammation, and if this was predictive of cardiac function. Male Sprague-Dawley rats underwent coronary artery ligation (30 min), followed by reperfusion to induce MI. [18F]LW223 PET was performed on d2, 7, 14 and 28. On d28, cardiac function was assessed by ultrasound. Naïve (n=10) and sham (n=6) rats were used as controls for comparison to the MI (n=5). A separate cohort of naïve (n=8), sham (n=18) and MI (n=17) rats were produced for histological validation. Troponin I measurements suggested a range of infarct severities (2583-10970ng/L). [18F]LW223 signal was highest within the MI cohort, and localised to the infarct. [18F]LW223 binding peaked at d2, with a smaller secondary peak within the infarct at d28. Manual counting by histology validated this pattern, and revealed that the majority of TSPO expressing cells within the infarct also expressed the monocyte/macrophage marker CD68 (55.2%). Finally, infarct [18F]LW223 signal at d2 correlated with infarct severity, and systolic dysfunction at d28. [18F]LW223 was able to map macrophage-driven inflammation in this model, with expression peaking on day 2, the extent of which was predictive of reduced cardiac function at day 28.

The 18 kDa translocator protein is a well-known biomarker of neuroinflammation, but also plays a role in homeostasis. PET with 18 kDa translocator protein radiotracers [C-11]PBR28 in humans and [F-18]GE180 in mice has demonstrated sex-dependent uptake patterns in the healthy brain, suggesting sex-dependent 18 kDa translocator protein expression, although humans and mice had differing results. This study aimed to assess whether the 18 kDa translocator protein PET radiotracer [F-18]LW223 exhibited sexually dimorphic uptake in healthy murine brain and peripheral organs. Male and female C57Bl6/J mice (13.6 +/- 5.4 weeks, 26.8 +/- 5.4 g, mean +/- SD) underwent 2 h PET scanning post-administration of [F-18]LW223 (6.7 +/- 3.6 MBq). Volume of interest and parametric analyses were performed using standard uptake values (90-120 min). Statistical differences were assessed by unpaired t-test or two-way ANOVA with & Scaron;idak's test (alpha = 0.05). The uptake of [F-18]LW223 was significantly higher across multiple regions of the male mouse brain, with the most pronounced difference detected in hypothalamus (P < 0.0001). Males also exhibited significantly higher [F-18]LW223 uptake in the heart when compared to females (P = 0.0107). Data support previous findings on sexually dimorphic 18 kDa translocator protein radiotracer uptake patterns in mice and highlight the need to conduct sex-controlled comparisons in 18 kDa translocator protein PET imaging studies.

Heterocyclic nonacetamide ligands are used as positron emission tomography (PET) imaging agents of the synaptic vesicle glycoprotein 2A (SV2A), with potential applications in the diagnosis of various neuropsychiatric diseases. To date, the main synthetic strategy to access these optically active compounds has involved the racemic synthesis of a late-stage intermediate followed by the separation of the enantiomers. Here, we describe the use of iminium organocatalysis for the asymmetric synthesis of SynVesT-1, an important PET imaging agent of SV2A. The key step involved the conjugate addition of nitromethane with a cinnamaldehyde in the presence of the Jørgensen-Hayashi catalyst using the Merck dual acid cocatalyst system. Pinnick-type oxidation and esterification of the adduct was then followed by chemoselective nitro group reduction and cyclization using nickel borate. N-Alkylation of the resulting lactam then completed the seven-step synthesis of SynVesT-1. This approach was amenable for the synthesis of an organotin analogue, which following copper(II)-mediated fluoro-destannylation allowed rapid access to [18F]SynVesT-1.

The positron emission tomography imaging agents cis- and trans-4-[18F]fluoro-l-proline are used for the detection of numerous diseases such as pulmonary fibrosis and various carcinomas. These imaging agents are typically prepared by nucleophilic fluorination of 4-hydroxy-l-proline derivatives, with [18F]fluoride, followed by deprotection. Although effective radiofluorination reactions have been developed, the overall radiosynthesis process is suboptimal due to deprotection methods that are performed manually, require multiple steps, or involve harsh conditions. Here we describe the development of two synthetic routes that allow access to precursors, which undergo highly selective radiofluorination reactions and rapid deprotection, under mild acidic conditions. These methods were found to be compatible with automation, avoiding manual handling of radioactive intermediates.

Purpose To provide a comprehensive assessment of the novel 18 kDa translocator protein (TSPO) radiotracer, [ 18 F]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 [ 18 F]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 ( V T ) and binding potential transfer corrected ( BP TC ) 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 [ 18 F]LW223 uptake kinetics in the brain and heart required the use of different outcome measures, with BP TC more stable in the heart and V T more stable in the brain. Conclusion Modelling of [ 18 F]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.

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 18 F-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: [ 18 F]FDG, [ 18 F]AlF-NOTA-RGDfK, [ 18 F]AlF-NOTA-octreotide ([ 18 F]AlF-NOTA-OC), [ 18 F]AlF-NOTA-NOC, [ 18 F]ENC2015 and [ 18 F]ENC2018. Filtered-back projection (FBP) and iterative methods were used for reconstruction of PET data. Predicted and true clinical absorbed doses for [ 18 F]FDG and [ 18 F]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.

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.

IntroductionPositron 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.Methods[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.Results[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.ConclusionQuantification 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.

The synthesis of a new class of benzotriazole-derived α-amino acid is described using a highly efficient nucleophilic aromatic substitution of ortho-fluoronitrobenzenes with l-3-aminoalanine and a polymer-supported nitrite reagent-mediated diazotization and cyclization of the subsequent 1,2-aryldiamines as the key steps. Further functionalization of the benzotriazole unit by preparation of halogenated analogues and Suzuki-Miyaura cross-coupling with aryl boronic acids allowed the synthesis of α-amino acids with conjugated side chains. Analysis of the photophysical properties of these α-amino acids revealed that incorporation of electron-rich substituents results in charge-transfer-based, fluorescent compounds with MegaStokes shifts.