Preclinical Positron Emission Tomography Studies Research Articles

Improved Chemical and Radiochemical Synthesis of Neuropeptide Y Y2 Receptor Antagonist N-Methyl-JNJ-31020028 and Preclinical Positron Emission Tomography Studies.

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals and is involved in several physiological processes through NPY Y1, Y2, Y4 and Y5 receptors. Of those, the Y2 receptor has particular relevance for its autoreceptor role in inhibiting the release of NPY and other neurotransmitters and for its involvement in relevant mechanisms such as feeding behaviour, cognitive processes, emotion regulation, circadian rhythms and disorders such as epilepsy and cancer. PET imaging of the Y2 receptor can provide a valuable platform to understand this receptor's functional role and evaluate its potential as a therapeutic target. In this work, we set out to refine the chemical and radiochemical synthesis of the Y2 receptor antagonist N-[11C]Me-JNJ31020028 for in vivo PET imaging studies. The non-radioactive reference compound, N-Me-JNJ-31020028, was synthesised through batch synthesis and continuous flow methodology, with 43% and 92% yields, respectively. N-[11C]Me-JNJ-31020028 was obtained with a radiochemical purity > 99%, RCY of 31% and molar activity of 156 GBq/μmol. PET imaging clearly showed the tracer's biodistribution in several areas of the mouse brain and gut where Y2 receptors are known to be expressed.

Reconstruction of multi-animal PET acquisitions with anisotropically variant PSF

Among other factors such as random, attenuation and scatter corrections, uniform spatial resolution is key to performing accurate quantitative studies in Positron emission tomography (PET). Particularly in preclinical PET studies involving simultaneous acquisition of multiple animals, the degradation of image resolution due to the depth of interaction (DOI) effect far from the center of the Field of View (FOV) becomes a significant concern. In this work, we incorporated a spatially-variant resolution model into a real time iterative reconstruction code to obtain accurate images of multi-animal acquisition. We estimated the spatially variant point spread function (SV-PSF) across the FOV using measurements and Monte Carlo (MC) simulations. The SV-PSF obtained was implemented in a GPU-based Ordered subset expectation maximization (OSEM) reconstruction code, which includes scatter, attenuation and random corrections. The method was evaluated with acquisitions from two preclinical PET/CT scanners of the SEDECAL Argus family: a Derenzo phantom placed 2 cm off center in the 4R-SuperArgus, and a multi-animal study with 4 mice in the 6R-SuperArgus. The SV-PSF reconstructions showed uniform spatial resolution without significant increase in reconstruction time, with superior image quality compared to the uniform PSF model.

Automated radiosynthesis of 1-(2-[18 F]fluoroethyl)-L-tryptophan ([18 F]FETrp) for positron emission tomography (PET) imaging of cancer in humans.

The radiotracer 1-(2-[18 F]fluoroethyl)-L-tryptophan (L-[18 F]FETrp or [18 F]FETrp) is a substrate of indoleamine 2,3-dioxygenase, the initial and key enzyme of the kynurenine pathway associated with tumoral immune resistance. In preclinical positron emission tomography studies, [18 F]FETrp is highly accumulated in a wide range of primary and metastatic cancers, such as lung cancer, prostate cancer, and gliomas. However, the clinical translation of this radiotracer into the first-in-human trial has not been reported, partially due to its racemization during radiofluorination which renders the purification of the final product challenging. However, efficient purification is essential for human studies in order to assure radiochemical and enantiomeric purity. In this work, we report a fully automated radiosynthesis of [18 F]FETrp on a Synthra RNPlus research module, including a one-pot two steps radiosynthesis, dual independent chiral and reverse-phase semipreparative high-performance liquid chromatography purifications, and solid-phase extraction-assisted formulation. The presented approach has led to its Investigational New Drug application and approval that allows the testing of this tracer in humans.

Aluminum Fluoride-18 Labeled Mannosylated Dextran: Radiosynthesis and Initial Preclinical Positron Emission Tomography Studies

PurposeIn addition to being expressed on liver sinusoidal endothelial cells, mannose receptors are also found on antigen-presenting cells, including macrophages, which are mainly involved in the inflammation process. Dextran derivatives of various sizes containing cysteine and mannose moieties have previously been labeled with 99mTc and used for single-photon emission computed tomography imaging of sentinel lymph nodes. In this study, we radiolabeled 21.3-kDa D10CM with positron-emitting 18F for initial positron emission tomography (PET) studies in rats.ProceduresD10CM was conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator and radiolabeled with the aluminum fluoride-18 method. The whole-body distribution kinetics and stability of the intravenously administered tracer were studied in healthy male Sprague-Dawley rats by in vivo PET/CT imaging, ex vivo gamma counting, and high-performance liquid chromatography analysis.ResultsAl[18F]F-NOTA-D10CM was obtained with a radiochemical purity of >99% and molar activity of 9.9 GBq/μmol. At 60 minutes after injection, an average of 84% of the intact tracer was found in the blood, indicating excellent in vivo stability. The highest radioactivity concentration was seen in the liver, spleen, and bone marrow, in which mannose receptors are highly expressed under physiological conditions. The uptake specificity was confirmed with in vivo blocking experiments.ConclusionsOur results imply that Al[18F]F-NOTA-D10CM is a suitable tracer for PET imaging. Further studies in disease models with mannose receptor CD206-positive macrophages are warranted to clarify the tracer’s potential for imaging of inflammation.Graphical abstract

18F]Atorvastatin Pharmacokinetics and Biodistribution in Healthy Female and Male Rats.

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that are widely used to prevent cardiovascular diseases. However, a series of pleiotropic mechanisms have been associated with statins, particularly with atorvastatin. Therefore, the assessment of [18F]atorvastatin kinetics with positron emission tomography (PET) may elucidate the mechanism of action of statins and the impact of sexual dimorphism, which is one of the most debated interindividual variations influencing the therapeutic efficacy. [18F]Atorvastatin was synthesized via a previously optimized 18F-deoxyfluorination strategy, used for preclinical PET studies in female and male Wistar rats (n = 7 for both groups), and for subsequent ex vivo biodistribution assessment. PET data were fitted to several pharmacokinetic models, which allowed for estimating relevant kinetic parameters. Both PET imaging and biodistribution studies showed negligible uptake of [18F]atorvastatin in all tissues compared with the primary target organ (liver), excretory pathways (kidneys and small intestine), and stomach. Uptake of [18F]atorvastatin was 38 ± 3% higher in the female liver than in the male liver. The irreversible 2-tissue compartment model showed the best fit to describe [18F]atorvastatin kinetics in the liver. A strong correlation (R2 > 0.93) between quantitative Ki (the radiotracer’s unidirectional net rate of influx between compartments) and semi-quantitative liver’s SUV (standard uptake value), measured between 40 to 90 min, showed potential to use the latter parameter, which circumvents the need for blood sampling as a surrogate of Ki for monitoring [18F]atorvastatin uptake. Preclinical assays showed faster uptake and clearance for female rats compared to males, seemingly related to a higher efficiency for exchanges between the arterial input and the hepatic tissue. Due to the slow [18F]atorvastatin kinetics, equilibrium between the liver and plasma concentration was not reached during the time frame studied, making it difficult to obtain sufficient and accurate kinetic information to quantitatively characterize the radiotracer pharmacokinetics over time. Nevertheless, the reported results suggest that the SUV can potentially be used as a simplified measure, provided all scans are performed at the same time point. Preclinical PET-studies with [18F]atorvastatin showed faster uptake and clearance in female compared to male rats, apparently related to higher efficiency for exchange between arterial blood and hepatic tissue.

Preclinical PET Studies of [11C]UCB-J Binding in Minipig Brain.

Loss of neuronal synapse function is associated with a number of brain disorders. The [11C]UCB-J positron emission tomography (PET) tracer allows for in vivo examination of synaptic density, as it binds to synaptic vesicle glycoprotein 2A (SV2A) expressed in presynaptic terminals. Here, we characterise [11C]UCB-J imaging in Göttingen minipigs. Using PET imaging, we examined tracer specificity and compared kinetic models. We explored the use of a standard blood curve and centrum semiovale white matter as a reference region. We compared in vivo [11C]UCB-J PET imaging to in vitro autoradiography, Western blotting and real-time quantitative polymerase chain reaction. The uptake kinetics of [11C]UCB-J could be described using a 1-tissue compartment model and blocking of SV2A availability with levetiracetam showed dose-dependent specific binding. Population-based blood curves resulted in reliable [11C]UCB-J binding estimates, while it was not possible to use centrum semiovale white matter as a non-specific reference region. Brain [11C]UCB-J PET signals correlated well with [3H]UCB-J autoradiography and SV2A protein levels. [11C]UCB-J PET is a valid in vivo marker of synaptic density in the minipig brain, with binding values close to those reported for humans. Minipig models of disease could be valuable for investigating the efficacy of putative neuroprotective agents for preserving synaptic function in future non-invasive, longitudinal studies.

Synthesis and Evaluation of 11C- and 18F-Labeled SOAT1 Inhibitors as Macrophage Foam Cell Imaging Agents.

PD-132301, an inhibitor of sterol O-acyltransferase 1 (SOAT1; also known as acyl-coenzyme A:cholesterol acyltransferase-1, ACAT1), is under clinical investigation for numerous adrenal disorders. Radiolabeled SOAT1 inhibitors could support drug discovery and help diagnose SOAT1-related disorders, such as atherosclerosis. We synthesized two radiolabeled SOAT1 inhibitors, [11C]PD-132301 and fluorine analogue [18F]1. Rat biodistribution studies were conducted with both agents and, as the most selective tracer, [11C]PD-132301 was advanced to preclinical positron emission tomography studies in (atherosclerotic) ApoE-/- mice. The uptake of [11C]PD-132301 in SOAT1-rich tissue warrants further investigation into the compound as an atherosclerosis and adrenal imaging agent.

Synthesis, Radiolabeling, and in Vitro and in Vivo Evaluation of [18F]ENL30: A Potential PET Radiotracer for the 5-HT7 Receptor

The 5-HT 7 receptor (5-HT 7 R) is involved in a broad range of physiological conditions and disorders. Currently, there is no validated clinical positron emission tomography (PET) tracer available; however, we have recently developed a promising 11 C-labeled candidate. In this project, we aimed to further extend our efforts and develop an 18 F-labeled derivative, coined [ 18 F]ENL30. Fluorine-18 has several advantages over carbon-11 especially within the preclinical phase, where a long half-life usually increases evaluation throughput. ENL30 was successfully synthesized in a low albeit sufficient overall yield. Radiolabeling succeeded with a radiochemical yield of approximately 4.5%. Subsequent preclinical PET studies revealed that [ 18 F]ENL30 binds specifically to the 5-HT 7 R but suffered from affinity to σ-receptors. Additionally, we identified [ 18 F]ENL30 to be a P-gp substrate in rats. However, we believe that [ 18 F]ENL30 may prove to be valuable in higher species that exhibit decreased P-gp dependency. If required, σ-receptor binding could, in such studies, be selectively blocked potentially allowing for selective 5-HT 7 R imaging.

Preparation and in vivo characterization of 51MnCl2 as PET tracer of Ca2+ channel-mediated transport

Manganese has long been employed as a T1-shortening agent in magnetic resonance imaging (MRI) applications, but these techniques are limited by the biotoxicity of bulk-manganese. Positron emission tomography (PET) offers superior contrast sensitivity compared with MRI, and recent preclinical PET studies employing 52gMn (t1/2: 5.6 d, β+: 29%) show promise for a variety of applications including cell tracking, neural tract tracing, immunoPET, and functional β-cell mass quantification. The half-life and confounding gamma emissions of 52gMn are prohibitive to clinical translation, but the short-lived 51Mn (t1/2: 46 min, β+: 97%) represents a viable alternative. This work develops methods to produce 51Mn on low-energy medical cyclotrons, characterizes the in vivo behavior of 51MnCl2 in mice, and performs preliminary human dosimetry predictions. 51Mn was produced by proton irradiation of electrodeposited isotopically-enriched 54Fe targets. Radiochemically isolated 51MnCl2 was intravenously administered to ICR mice which were scanned by dynamic and static PET, followed by ex vivo gamma counting. Rapid blood clearance was observed with stable uptake in the pancreas, kidneys, liver, heart, and salivary gland. Dosimetry calculations predict that 370 MBq of 51Mn in an adult human male would yield an effective dose equivalent of approximately 13.5 mSv, roughly equivalent to a clinical [18F]-FDG procedure.

Effects of isoflurane anesthesia and intravenous morphine self-administration on regional glucose metabolism ([18 F]FDG-PET) of male Sprague-Dawley rats.

Although certain drugs of abuse are known to disrupt brain glucose metabolism (BGluM), the effects of opiates on BGluM are not well characterized. Moreover, preclinical positron emission tomography (PET) studies anesthetize animals during the scan, which limits clinical applications. We investigated the effects of (i) isoflurane anesthesia and (ii) intravenous morphine self-administration (MSA) on BGluM in rats. Jugular vein cannulated adult male Sprague-Dawley rats self-administered either saline (SSA) or morphine (0.5mg/kg/infusion, 4h/day for 12days). All animals were scanned twice with [18 F]-fluoro-deoxy-glucose (FDG)-PET/CT at a baseline and at 2-day withdrawal from self-administration. After the IV injection of FDG, one batch of animals (n=14) was anesthetized with isoflurane and the other batch (n=16) was kept awake during the FDG uptake (45min). After FDG uptake, all animals were anesthetized in order to perform a PET/CT scan (30min). Isoflurane anesthesia, as compared to the awake condition, reduced BGluM in the olfactory, cortex, thalamus, and basal ganglia, while increasing BGluM in the midbrain, hypothalamus, hippocampus, and cerebellum. Morphine self-administered animals exhibited withdrawal signs (piloerection and increased defecation), drug seeking, and locomotor stimulation to morphine (0.5mg/kg) during the 2day withdrawal. The BGluM in the striatum was increased in the MSA group as compared to the SSA group; this effect was observed only in the isoflurane anesthesia, not the awake condition. These findings suggest that the choice of the FDG uptake condition may be important in preclinical PET studies and increased BGluM in the striatum may be associated with opiate seeking in withdrawal.

Markerless rat head motion tracking using structured light for brain PET imaging of unrestrained awake small animals

Preclinical positron emission tomography (PET) imaging in small animals is generally performed under anesthesia to immobilize the animal during scanning. More recently, for rat brain PET studies, methods to perform scans of unrestrained awake rats are being developed in order to avoid the unwanted effects of anesthesia on the brain response. Here, we investigate the use of a projected structure stereo camera to track the motion of the rat head during the PET scan. The motion information is then used to correct the PET data. The stereo camera calculates a 3D point cloud representation of the scene and the tracking is performed by point cloud matching using the iterative closest point algorithm. The main advantage of the proposed motion tracking is that no intervention, e.g. for marker attachment, is needed. A manually moved microDerenzo phantom experiment and 3 awake rat [18F]FDG experiments were performed to evaluate the proposed tracking method. The tracking accuracy was 0.33 mm rms. After motion correction image reconstruction, the microDerenzo phantom was recovered albeit with some loss of resolution. The reconstructed FWHM of the 2.5 and 3 mm rods increased with 0.94 and 0.51 mm respectively in comparison with the motion-free case. In the rat experiments, the average tracking success rate was 64.7%. The correlation of relative brain regional [18F]FDG uptake between the anesthesia and awake scan reconstructions was increased from on average 0.291 (not significant) before correction to 0.909 (p < 0.0001) after motion correction. Markerless motion tracking using structured light can be successfully used for tracking of the rat head for motion correction in awake rat PET scans.

Preclinical imaging evaluation of novel TSPO-PET ligand 2-(5,7-Diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ([ (18)F]VUIIS1008) in glioma.

Translocator protein (TSPO) concentrations are elevated in glioma, suggesting a role for TSPO positron emission tomography (PET) imaging in this setting. In preclinical PET studies, we evaluated a novel, high-affinity TSPO PET ligand, [(18)F]VUIIS1008, in healthy mice and glioma-bearing rats. Dynamic PET data were acquired simultaneously with [(18)F]VUIIS1008 injection, with binding reversibility and specificity evaluated in vivo by non-radioactive ligand displacement or blocking. Compartmental analysis of PET data was performed using metabolite-corrected arterial input functions. Imaging was validated with histology and immunohistochemistry. [(18)F]VUIIS1008 exhibited rapid uptake in TSPO-rich organs. PET ligand uptake was displaceable with non-radioactive VUIIS1008 or PBR06 in mice. Tumor accumulation of [(18)F]VUIIS1008 was blocked by pretreatment with VUIIS1008 in rats. [(18)F]VUIIS1008 exhibited improved tumor-to-background ratio and higher binding potential in tumors compared to a structurally similar pyrazolopyrimidine TSPO ligand, [(18)F]DPA-714. The PET ligand [(18)F]VUIIS1008 exhibits promising characteristics as a tracer for imaging glioma. Further translational studies appear warranted.

Analysis of 5-HT2A Receptor Binding with [11C]MDL 100907 in Rats: Optimization of Kinetic Modeling

Preclinical positron emission tomography studies are important to follow disease progression and develop new pharmacological agents. We investigated whether kinetic modeling of 5-HT2A tracer [(11)C]MDL 100907 is possible in rats. Kinetic modeling with either metabolite-corrected plasma curve or with the cerebellum as a reference tissue resulted in a good correlation of nondisplaceable binding potential (BPND) calculated from a two-tissue compartment model (2TCM) or different reference tissue models. Injecting the tracer by a slower bolus decreases the variation in 2TCM outcome parameters and results in a good correlation between k3/k4 and the other models. Application of 0.2mg/kg cold MDL 100907 resulted in almost complete occupancy of 5-HT2A receptors. A reference tissue model can be used for [(11)C]MDL kinetic modeling in rats, which is preferable in pharmacological or longitudinal studies.

Usefulness of Automatic Quantification of Immunochemical Staining on Whole Tumor Sections for Correlation with Oncological Small Animal PET Studies: An Example with Cell Proliferation, Glucose Transporter 1 and FDG

To highlight the use of automatic quantification of immunochemical staining on digitized images of whole tumor sections in preclinical positron emission tomography (PET) studies. Xenografted human testicular tumors (36) were imaged with 2-deoxy-2[F-18]fluoro-D: -glucose (FDG) small animal PET (SA-PET). Tumor cell proliferation and glucose transportation were assessed with cyclin A and Glut-1 immunostaining. Tumor slides were digitized and processed with PixCyt software enabling whole slide quantification, then compared with junior and senior pathologist manual scoring. Manual and automatic quantification results were correlated to FDG uptake. For cyclin A, inter- and intra-observer agreement for manual scoring was 0.52 and 0.72 and concordance between senior pathologist and automatic quantification was 0.84. Correlations between Tumor/Background ratio and tumor cell proliferation assessed by automatic quantification, junior and senior pathologists were 0.75, 0.55, and 0.61, respectively. Correlation between Tumor/Background ratio and Glut-1 assessed by automatic quantification was 0.74. Automatic quantification of immunostaining is a valuable tool to overcome inter- and intra-observer variability for correlation of cell proliferation or other markers with tumor tracer uptake.