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Abstract Background The demand for 68Ga-labeled radiotracers has significantly increased in the past decade, driven by the development of diversified imaging tracers, such as FAPI derivatives, PSMA-11, DOTA-TOC, and DOTA-TATE. These tracers have exhibited promising results in theranostic applications, fueling interest in exploring them for clinical use. Among these probes, 68Ga-labeled FAPI-46 and DOTA-TOC have emerged as key players due to their ability to diagnose a broad spectrum of cancers ([68Ga]Ga-FAPI-46) in late-phase studies, whereas [68Ga]Ga-DOTA-TOC is clinically approved for neuroendocrine tumors. To facilitate their production, we leveraged a microfluidic cassette-based iMiDEV radiosynthesizer, enabling the synthesis of [68Ga]Ga-FAPI-46 and [68Ga]Ga-DOTA-TOC based on a dose-on-demand (DOD) approach. Results Different mixing techniques were explored to influence radiochemical yield. We achieved decay-corrected yield of 44 ± 5% for [68Ga]Ga-FAPI-46 and 46 ± 7% for [68Ga]Ga-DOTA-TOC in approximately 30 min. The radiochemical purities (HPLC) of [68Ga]Ga-FAPI-46 and [68Ga]Ga-DOTA-TOC were 98.2 ± 0.2% and 98.4 ± 0.9%, respectively. All the quality control results complied with European Pharmacopoeia quality standards. We optimized various parameters, including 68Ga trapping and elution, cassette batches, passive mixing in the reactor, and solid-phase extraction (SPE) purification and formulation. The developed synthesis method reduced the amount of precursor and other chemicals required for synthesis compared to conventional radiosynthesizers. Conclusions The microfluidic-based approach enabled the implementation of radiosynthesis of [68Ga]Ga-FAPI-46 and [68Ga]Ga-DOTA-TOC on the iMiDEV™ microfluidic module, paving the way for their use in preclinical and clinical applications. The microfluidic synthesis approach utilized 2–3 times less precursor than cassette-based conventional synthesis. The synthesis method was also successfully validated in a similar microfluidic iMiDEV module at a different research center for the synthesis of [68Ga]Ga-FAPI-46 with limited runs. Our study demonstrated the potential of microfluidic methods for efficient and reliable radiometal-based radiopharmaceutical synthesis, contributing valuable insights for future advancements in this field and paving the way for routine clinical applications in the near future.

Abstract A positron emission tomography (PET) tracer detecting α-synuclein pathology will improve the diagnosis, and ultimately the treatment of α-synuclein-related diseases. Here we show that the PET ligand, [18F]ACI-12589, displays good in vitro affinity and specificity for pathological α-synuclein in tissues from patients with different α-synuclein-related disorders including Parkinson’s disease (PD) and Multiple-System Atrophy (MSA) using autoradiography and radiobinding techniques. In the initial clinical evaluation we include 23 participants with α-synuclein related disorders, 11 with other neurodegenerative disorders and eight controls. In vivo [18F]ACI-12589 demonstrates clear binding in the cerebellar white matter and middle cerebellar peduncles of MSA patients, regions known to be highly affected by α-synuclein pathology, but shows limited binding in PD. The binding statistically separates MSA patients from healthy controls and subjects with other neurodegenerative disorders, including other synucleinopathies. Our results indicate that α-synuclein pathology in MSA can be identified using [18F]ACI-12589 PET imaging, potentially improving the diagnostic work-up of MSA and allowing for detection of drug target engagement in vivo of novel α-synuclein targeting therapies.

Bruton’s tyrosine kinase (BTK) is pivotal in B-cell signaling and a target for potential anti-cancer and immunological disorder therapies. Improved selective reversible BTK inhibitors are in demand due to the absence of direct BTK engagement measurement tools. Promisingly, PET imaging can non-invasively evaluate BTK expression. In this study, radiolabeled BIO-2008846 ([11C]BIO-2008846-A), a BTK inhibitor, was used for PET imaging in NHPs to track brain biodistribution. Radiolabeling BIO-2008846 with carbon-11, alongside four PET scans on two NHPs each, showed a homogeneous distribution of [11C]BIO-2008846-A in NHP brains. Brain uptake ranged from 1.8% ID at baseline to a maximum of 3.2% post-pretreatment. The study found no significant decrease in regional VT values post-dose, implying minimal specific binding of [11C]BIO-2008846-A compared to free and non-specific components in the brain. Radiometabolite analysis revealed polar metabolites with 10% unchanged radioligand after 30 min. The research highlighted strong brain uptake despite minor distribution variability, confirming passive diffusion kinetics dominated by free and non-specific binding.

Abstract Background Correlations between dopamine transporter (DAT) availability and Parkinson’s disease (PD) motor symptoms vary depending on the imaging modality, choice of regions of interest and clinical measures. We aimed to validate the PET radioligand [18F]FE-PE2I as a clinical biomarker in PD, hypothesizing negative correlations between DAT availability in specified nigrostriatal regions with symptom duration, disease stage and motor symptom scores. Methods We included 41 PD patients (age 45–79 years; H&Y stage

Microfluidic technology is a highly efficient technique used in positron emission tomography (PET) radiochemical synthesis. This approach enables the precise control of reactant flows and reaction conditions, leading to improved yields and reduced synthesis time. The synthesis of two radiotracers, L-[11C]methionine and [11C]choline, was performed, using a microfluidic cassette and an iMiDEVTM module by employing a dose-on-demand approach for the synthesis process. We focused on optimizing the precursor amounts and radiosynthesis on the microfluidic cassette. L-[11C]methionine and [11C]choline were synthesized using a microreactor filled with a suitable resin for the radiochemical reaction. Trapping of the [11C]methyl iodide, its reaction, and solid-phase extraction purification were performed on a microreactor, achieving radiochemical yields of >80% for L-[11C]methionine and >60% for [11C]choline (n = 3). The total synthesis time for both the radiotracers was approximately 20 min. All quality control tests complied with the European Pharmacopeia standards. The dose-on-demand model allows for real-time adaptation to patient schedules, making it suitable for preclinical and clinical settings. Precursor optimization enhanced the cost efficiency without compromising the yield. The importance of dose-on-demand synthesis and optimized precursor utilization to produce L-[11C]methionine and [11C]choline was emphasized in this study. The results demonstrated the feasibility of dose-on-demand adaptations for clinical applications with reduced precursor quantities and high radiochemical yields.

The aim of this study was to measure the brain penetrance and kinetics of BIIB104, a first-in-class AMPA receptor potentiator developed for cognitive impairment associated with schizophrenia. It was recently halted in phase 2 clinical development, and there are a lack of tools to directly measure AMPA receptor engagement. To achieve this, the drug candidate was radiolabeled with carbon-11, and its brain penetrance and kinetics were measured in non-human primates via dynamic PET scans. Radiolabeling was achieved through a three-step nucleophilic [11C]cyanation reaction in one pot, resulting in the high radioactivity and radiochemical purity (>99%) of [11C]BIIB104. The study found that [11C]BIIB104 entered the non-human primate brains at 4–5% ID at peak, with a homogeneous distribution. However, a mild regional heterogeneity was observed in the thalamus. The lack of conclusive evidence for a change in regional values after BIIB104 dosing suggests that any specific binding component of BIIB104 is negligible compared to the free and non-specific components in the living brain. Overall, the study demonstrated high brain uptake with minor variability in [11C]BIIB104 distribution across various brain regions, its kinetics were consistent with those of passive diffusion, and the dominating components were the free concentration and non-specific binding. This information is valuable for understanding the potential effects and mechanisms of BIIB104 in the brain.

Abstract Background Monoacylglycerol lipase (MAGL) is a key serine hydrolase which terminates endocannabinoid signaling and regulates arachidonic acid driven inflammatory responses within the central nervous system. To develop [11C]PF-06809247 into a clinically usable MAGL positron emission tomography (PET) radioligand, we assessed the occupancy of MAGL by an inhibitor in the non-human primate (NHP) brain. Additionally, we measured the whole-body distribution of [11C]PF-06809247 in NHP and estimated human effective radiation doses. Methods Seven cynomolgus monkeys were enrolled for brain PET measurements. Two PET measurements along with arterial blood sampling were performed in each NHP: one baseline and one pretreatment condition with intravenous administration of PF-06818883, a pro-drug of a selective MAGL inhibitor (total of seven doses between 0.01 and 1.27 mg/kg). Kinetic parameters K 1, k 2 and k 3 were estimated by a two tissue compartment (2TC) model using metabolite corrected plasma radioactivity as the input function. k 4 was set as 0 according to the irreversible binding of [11C]PF-06809247. K i by 2TC and Patlak analysis were calculated as the influx constant. The target occupancy was calculated using K i at baseline and pretreatment conditions. Two cynomolgus monkeys were enrolled for whole-body PET measurements. Estimates of the absorbed radiation dose in humans were calculated with OLINDA/EXM 1.1 using the adult male reference model. Results Radioactivity retention was decreased in all brain regions following pretreatment with PF-06818883. Occupancy was measured as 25.4–100.5% in a dose dependent manner. Whole-body PET showed high radioactivity uptake values in the liver, small intestine, kidney, and brain. The effective dose of [11C]PF-06809247 was calculated as 4.3 μSv/MBq. Conclusions [11C]PF-06809247 is a promising PET ligand for further studies of MAGL in the human brain.

Muscarinic acetylcholine receptors (mAChR), including M4, draw attention as therapeutic targets for several neurodegenerative diseases including Alzheimer’s disease (AD). PET imaging of M4 positive allosteric modulator (PAM) allows qualification of the distribution as well as the expression of this receptor under physiological conditions and thereby helps to assess the receptor occupancy (RO) of a drug candidate. In this study, our aims were (a) to synthesize a novel M4 PAM PET radioligand [11C]PF06885190 (b) to evaluate the brain distribution of [11C]PF06885190 in nonhuman primates (NHP) and (c) to analyze its radiometabolites in the blood plasma of NHP. Radiolabeling of [11C]PF06885190 was accomplished via N-methylation of the precursor. Six PET measurements were performed using two male cynomolgus monkeys, where three PET measurements were at baseline, two after pretreatment with a selective M4 PAM compound CVL-231 and one after pretreatment with donepezil. The total volume of distribution (VT) of [11C]PF06885190 was examined using Logan graphical analysis with arterial input function. Radiometabolites were analyzed in monkey blood plasma using gradient HPLC system. Radiolabeling of [11C]PF06885190 was successfully accomplished and the radioligand was found to be stable in the formulation, with radiochemical purity exceeding 99% 1 h after the end of the synthesis. [11C]PF06885190 was characterized in the cynomolgus monkey brain where a moderate brain uptake was found at the baseline condition. However, it showed fast wash-out as it dropped to half of the peak at around 10 min. Change of VT from baseline was around −10% after pretreatment with a M4 PAM, CVL-231. Radiometabolite studies showed relatively fast metabolism. Although sufficient brain uptake of [11C]PF06885190 was observed, these data suggest that [11C]PF06885190 might have too low specific binding in the NHP brain to be further applied in PET imaging.

Abstract Molecular imaging studies have shown low cerebral concentration of serotonin transporter in patients suffering from depression, compared to healthy control subjects. Whether or not this difference also is present before disease onset and after remission (i.e. a trait), or only at the time of the depressive episode (i.e. a state) remains to be explored. We examined 17 patients with major depressive disorder with positron emission tomography using [11C]MADAM, a radioligand that binds to the serotonin transporter, before and after treatment with internet-based cognitive behavioral therapy. In all, 17 matched healthy control subjects were examined once. Cerebellum was used as reference to calculate the binding potential. Differences before and after treatment, as well as between patients and controls, were assessed in a composite cerebral region and in the median raphe nuclei. All image analyses and confirmatory statistical tests were preregistered. Depression severity decreased following treatment (p

Abstract Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that mediates the retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Mutations such as D620N in the VPS35 gene have been identified in patients with autosomal dominant Parkinson’s disease (PD). However, it remains poorly understood whether and how VPS35 deficiency or mutation contributes to PD pathogenesis; specifically, the studies that have examined VPS35 thus far have differed in results and methodologies. We generated a VPS35 D620N mouse model using a Rosa26-based transgene expression platform to allow expression in a spatial manner, so as to better address these discrepancies. Here, aged (20-months-old) mice were first subjected to behavioral tests. Subsequently, DAB staining analysis of substantia nigra (SN) dopaminergic neurons with the marker for tyrosine hydroxylase (TH) was performed. Next, HPLC was used to determine dopamine levels, along with levels of its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum. Western blotting was also performed to study the levels of key proteins associated with PD. Lastly, autoradiography (ARG) evaluation of [3H]FE-PE2I binding to the striatal dopamine transporter DAT was carried out. We found that VPS35 D620N Tg mice displayed a significantly higher dopamine level than NTg counterparts. All results were then compared with that of current VPS35 studies to shed light on the disease pathogenesis. Our model allows future studies to explicitly control spatial expression of the transgene which would generate a more reliable PD phenotype.

Abstract Positron emission tomography (PET) offers the study of biochemical, physiological, and pharmacological functions at a cellular and molecular level. The performance of a PET study mostly depends on the used radiotracer of interest. However, the development of a novel PET tracer is very difficult, as it is required to fulfill a lot of important criteria. PET radiotracers usually encounter different chemical modifications including redox reaction, hydrolysis, decarboxylation, and various conjugation processes within living organisms. Due to this biotransformation, different chemical entities are produced, and the amount of the parent radiotracer is declined. Consequently, the signal measured by the PET scanner indicates the entire amount of radioactivity deposited in the tissue; however, it does not offer any indication about the chemical disposition of the parent radiotracer itself. From a radiopharmaceutical perspective, it is necessary to quantify the parent radiotracer’s fraction present in the tissue. Hence, the identification of radiometabolites of the radiotracers is vital for PET imaging. There are mainly two reasons for the chemical identification of PET radiometabolites: firstly, to determine the amount of parent radiotracers in plasma, and secondly, to rule out (if a radiometabolite enters the brain) or correct any radiometabolite accumulation in peripheral tissue. Besides, radiometabolite formations of the tracer might be of concern for the PET study, as the radiometabolic products may display considerably contrasting distribution patterns inside the body when compared with the radiotracer itself. Therefore, necessary information is needed about these biochemical transformations to understand the distribution of radioactivity throughout the body. Various published review articles on PET radiometabolites mainly focus on the sample preparation techniques and recently available technology to improve the radiometabolite analysis process. This article essentially summarizes the chemical and structural identity of the radiometabolites of various radiotracers including [11C]PBB3, [11C]flumazenil, [18F]FEPE2I, [11C]PBR28, [11C]MADAM, and (+)[18F]flubatine. Besides, the importance of radiometabolite analysis in PET imaging is also briefly summarized. Moreover, this review also highlights how a slight chemical modification could reduce the formation of radiometabolites, which could interfere with the results of PET imaging. Graphical abstract

Abstract Background Reliable quantification of dopamine transporter (DAT), a biomarker for Parkinson’s disease (PD), is essential for diagnostic purposes as well as for evaluation of potential disease-modifying treatment. Due to degeneration of dopaminergic neurons and thus lower expected radioligand binding to DAT, higher measurement variability in PD patients might be expected than earlier reproducibility results in healthy controls. Therefore, we aimed to examine the test-retest properties of [18F]FE-PE2I-PET in PD patients. Methods Nine patients with PD (Hoehn and Yahr stage < 3) were included (men/women 6/3; mean age 65.2 ± 6.8 years). Each patient underwent two [18F]FE-PE2I-PET measurements within 7–28 days. The outcome measure was non-displaceable binding potential generated using wavelet-aided parametric imaging with cerebellum as reference region. We assessed test-retest performance using estimates of reliability and repeatability. Regions for primary analysis were caudate, putamen, ventral striatum, and substantia nigra. Exploratory analysis was performed for functional subdivisions of the striatum. We also compared the more vs. less affected side. Results [18F]FE-PE2I showed absolute variability estimates of 5.3–7.6% in striatal regions and 11% in substantia nigra and ICCs of 0.74–0.97 (median 0.91). The absolute variability for functional striatal subdivisions was 6.0–9.6% and ICCs of 0.76–0.91 (median 0.91). The less affected substantia nigra exhibited greater consistency than the more affected side. According to power calculations based on the current sample size, DAT changes of 5–11% in the striatum and 28% in the substantia nigra can be detected with a power of 0.8 (p < 0.0125). Conclusion DAT-PET measurements with [18F]FE-PE2I in PD patients showed good repeatability and reliability. The slightly lower reliability in the substantia nigra in patients may be explained by lower DAT density and smaller anatomical size. Power calculations suggest that [18F]FE-PE2I PET is a suitable marker for longitudinal DAT decline in PD. Trial registration EudraCT 2017-003327-29

Abstract Positron emission tomography (PET) is a non‐invasive imaging method to measure the molecule in vivo. PET imaging can evaluate the central nervous system drugs as target engagement in the human brain. For antipsychotic drugs, adequate dopamine D2 receptor occupancy (“therapeutic window”) is reported to be from 65%‐70% to 80% to achieve the antipsychotic effect without extrapyramidal symptoms. For antidepressants, the clinical threshold of serotonin transporter (5‐HTT) occupancy is reported to be 70%‐80% although the relation between the side effect and 5‐HTT occupancy has not yet been established. Evaluation of norepinephrine transporter (NET) occupancy for antidepressant is ongoing as adequate PET radioligands for NET were developed recently. Measurement of the target occupancy has been a key element to evaluate the in vivo target engagement of the drugs. In order to evaluate new drug targets for disease conditions such as negative symptoms/cognitive impairment of schizophrenia and treatment‐resistant depression, new PET radioligands need to be developed concurrently with the drug development.

Abstract Background The radioligand [11C]VC-002 was introduced in a small initial study long ago for imaging of muscarinic acetylcholine receptors (mAChRs) in human lungs using positron emission tomography (PET). The objectives of the present study in control subjects were to advance the methodology for quantification of [11C]VC-002 binding in lung and to examine the reliability using a test-retest paradigm. This work constituted a self-standing preparatory step in a larger clinical trial aiming at estimating mAChR occupancy in the human lungs following inhalation of mAChR antagonists. Methods PET measurements using [11C]VC-002 and the GE Discovery 710 PET/CT system were performed in seven control subjects at two separate occasions, 2–19 days apart. One subject discontinued the study after the first measurement. Radioligand binding to mAChRs in lung was quantified using an image-derived arterial input function. The total distribution volume (V T) values were obtained on a regional and voxel-by-voxel basis. Kinetic one-tissue and two-tissue compartment models (1TCM, 2TCM), analysis based on linearization of the compartment models (multilinear Logan) and image analysis by data-driven estimation of parametric images based on compartmental theory (DEPICT) were applied. The test-retest repeatability of V T estimates was evaluated by absolute variability (VAR) and intraclass correlation coefficients (ICCs). Results The 1TCM was the statistically preferred model for description of [11C]VC-002 binding in the lungs. Low VAR (< 10%) across analysis methods indicated good reliability of the PET measurements. The V T estimates were stable after 60 min. Conclusions The kinetic behaviour and good repeatability of [11C]VC-002 as well as the novel lung image analysis methodology support its application in applied studies on drug-induced mAChR receptor occupancy and the pathophysiology of pulmonary disorders. Trial registration ClinicalTrials.gov identifier: NCT03097380 , registered: 31 March 2017.

Abstract Introduction A rapid transition from a clinical‐based classification to a pathology‐based classification of neurodegenerative conditions, largely promoted by the increasing availability of imaging biomarkers, is emerging. The Framework for Innovative Multi‐tracer molecular Brain Imaging, funded by the EU Joint Program ‐ Neurodegenerative Disease Research 2016 “Working Groups for Harmonisation and Alignment in Brain Imaging Methods for Neurodegeneration,” aimed at providing a roadmap for the applications of established and new molecular imaging techniques in dementia. Methods We consider current and future implications of adopting a pathology‐based framework for the use and development of positron emission tomography techniques. Results This approach will enhance efforts to understand the multifactorial etiology of Alzheimer's disease and other dementias. Discussion The availability of pathology biomarkers will soon transform clinical and research practice. Crucially, a comprehensive understanding of strengths and caveats of these techniques will promote an informed use to take full advantage of these tools.

In the last decade, microfluidic techniques have been explored in radiochemistry, and some of them have been implemented in preclinical production. However, these are not suitable and reliable for preparing different types of radiotracers or dose-on-demand production. A fully automated iMiDEV™ microfluidic radiosynthesizer has been introduced and this study is aimed at using of the iMiDEV™ radiosynthesizer with a microfluidic cassette to produce [11C]flumazenil and [11C]L-deprenyl. These two are known PET radioligands for benzodiazepine receptors and monoamine oxidase-B (MAO-B), respectively. Methods were successfully developed to produce [11C]flumazenil and [11C]L-deprenyl using [11C]methyl iodide and [11C]methyl triflate, respectively. The final products 1644 ± 504 MBq (n = 7) and 533 ± 20 MBq (n = 3) of [11C]flumazenil and [11C]L-deprenyl were produced with radiochemical purities were over 98% and the molar activity for [11C]flumazenil and [11C]L-deprenyl was 1912 ± 552 GBq/µmol, and 1463 ± 439 GBq/µmol, respectively, at the end of synthesis. All the QC tests complied with the European Pharmacopeia. Different parameters, such as solvents, bases, methylating agents, precursor concentration, and different batches of cassettes, were explored to increase the radiochemical yield. Synthesis methods were developed using 3–5 times less precursor than conventional methods. The fully automated iMiDEV™ microfluidic radiosynthesizer was successfully applied to prepare [11C]flumazenil and [11C]L-deprenyl.

Abstract Background Over the last decade, a few radioligands have been developed for PET imaging of brain 5-HT1B receptors. The 5-HT1B receptor is a G-protein-coupled receptor (GPCR) that exists in two different agonist affinity states. An agonist ligand is expected to be more sensitive towards competition from another agonist, such as endogenous 5-HT, than an antagonist ligand. It is of interest to know whether the intrinsic activity of a PET radioligand for the 5-HT1B receptor impacts on its ability to detect changes in endogenous synaptic 5-HT density. Three high-affinity 11C-labeled 5-HT1B PET radioligands with differing intrinsic activity were applied to PET measurements in cynomolgus monkey to evaluate their sensitivity to be displaced within the brain by endogenous 5-HT. For these experiments, fenfluramine was pre-administered at two different doses (1.0 and 5.0 mg/kg, i.v.) to induce synaptic 5-HT release. Results A dose-dependent response to fenfluramine was detected for all three radioligands. At the highest dose of fenfluramine (5.0 mg/kg, i.v.), reductions in specific binding in the occipital cortex increased with radioligand agonist efficacy, reaching 61% for [11C]3. The most antagonistic radioligand showed the lowest reduction in specific binding. Conclusions Three 5-HT1B PET radioligands were identified with differing intrinsic activity that could be used in imaging high- and low-affinity states of 5-HT1B receptors using PET. From this limited study, radioligand sensitivity to endogenous 5-HT appears to depend on agonist efficacy. More extensive studies are required to substantiate this suggestion.

Abstract Background Positron emission tomography (PET) is a non-invasive molecular imaging technique that traces the distribution of radiolabeled molecules in experimental animals and human subjects. We hypothesized that PET could be used to visualize the binding of the bronchodilator drug ipratropium to muscarinic receptors (MR) in the lungs of living non-human primates (NHP). The objectives of this study were two-fold: (i) to develop a methodology for quantitative imaging of muscarinic receptors in NHP lung and (ii) to estimate and compare ipratropium-induced MR occupancy following drug administration via intravenous injection and inhalation, respectively. Methods A series of PET measurements (n = 18) was performed after intravenous injection of the selective muscarinic radioligand 11C-VC-002 in NHP (n = 5). The lungs and pituitary gland (both rich in MR) were kept in the field of view. Each PET measurement was followed by a PET measurement preceded by treatment with ipratropium (intravenous or inhaled). Results Radioligand binding was quantified using the Logan graphical analysis method providing the total volume of distribution (V T ). Ipratropium reduced the V T in the lung and pituitary in a dose-dependent fashion. At similar plasma ipratropium concentrations, administration by inhalation produced larger reductions in V T for the lungs. The plasma-derived apparent affinity for ipratropium binding in the lung was one order of magnitude higher after inhalation (K iih = 1.01 nM) than after intravenous infusion (K iiv = 10.84 nM). Conclusion Quantitative muscarinic receptor occupancy imaging by PET articulates and quantifies the therapeutic advantage of the inhaled route of delivery and provides a tool for future developments of improved inhaled drugs.

Abstract Background The G-protein-coupled receptor 44 (GPR44) is a beta cell-restricted target that may serve as a marker for beta cell mass (BCM) given the development of a suitable PET ligand. Methods The binding characteristics of the selected candidate, AZ12204657, at human GPR44 were determined using in vitro ligand binding assays. AZ12204657 was radiolabeled using 11C- or 3H-labeled methyl iodide ([11C/3H]CH3I) in one step, and the conversion of [11C/3H]CH3I to the radiolabeled product [11C/3H]AZ12204657 was quantitative. The specificity of radioligand binding to GPR44 and the selectivity for beta cells were evaluated by in vitro binding studies on pancreatic sections from human and non-human primates as well as on homogenates from endocrine and exocrine pancreatic compartments. Results The radiochemical purity of the resulting radioligand [11C]AZ12204657 was > 98%, with high molar activity (MA), 1351 ± 575 GBq/μmol (n = 18). The radiochemical purity of [3H]AZ12204657 was > 99% with MA of 2 GBq/μmol. Pancreatic binding of [11C/3H]AZ12204657 was co-localized with insulin-positive islets of Langerhans in non-diabetic individuals and individuals with type 2 diabetes (T2D). The binding of [11C]AZ12204657 to GPR44 was > 10 times higher in islet homogenates compared to exocrine homogenates. In human islets of Langerhans GPR44 was co-expressed with insulin, but not glucagon as assessed by co-staining and confocal microscopy. Conclusion We radiolabeled [11C]AZ12204657, a potential PET radioligand for the beta cell-restricted protein GPR44. In vitro evaluation demonstrated that [3H]AZ12204657 and [11C]AZ12204657 selectively target pancreatic beta cells. [11C]AZ12204657 has promising properties as a marker for human BCM.

Abstract Background The D1-dopamine receptor radioligand [11C]SCH23390 has been frequently used in PET studies. In drug-naïve patients with schizophrenia, the findings have been inconsistent, with decreases, increases, and no change in the frontal cortex D1-dopamine receptors. While these discrepancies are likely primarily due to a lack of statistical power in these studies, we speculated that an additional explanation may be the differences due to methods of image analysis between studies, affecting reliability as well as bias between groups. Methods Fifteen healthy subjects underwent two PET measurements with [11C]SCH23390 on the same day. The binding potential (BPND) was compared using a 95% confidence interval following manual and automated delineation of a region of interest (ROI) as well as with and without frame-by-frame realignment. Results Automated target region delineation produced lower BPND values, while automated delineation of the reference region yielded higher BPND values. However, no significant differences were observed for repeatability using automated and manual delineation methods. Frame-by-frame realignment generated higher BPND values and improved repeatability. Conclusions The results suggest that the choice of ROI delineation method is not an important factor for reliability, whereas the improved results following movement correction confirm its importance in PET image analysis. Realignment is therefore especially important for measurements in patient populations such as schizophrenia or Parkinson’s disease, where motion artifacts may be more prevalent.

Abstract The serotonin (5-HT) system plays an important role in the pathophysiology and treatment of several major psychiatric disorders. Currently, no suitable positron emission tomography (PET) imaging paradigm is available to assess 5-HT release in the living human brain. [11C]AZ10419369 binds to 5-HT1B receptors and is one of the most 5-HT-sensitive radioligands available. This study applied 5-HT concentration enhancers which can be safely studied in humans, and examined their effect on [11C]AZ10419369 binding at clinically relevant doses, including amphetamine (1 mg/kg), 3,4-methylenedioxymethamphetamine (MDMA; 1 mg/kg) or 5-hydroxy-L-tryptophan (5-HTP; 5 mg/kg). Twenty-six PET measurements (14 for amphetamine, 6 for MDMA and 6 for 5-HTP) using a bolus and constant infusion protocol were performed in four cynomolgus monkeys before or after drug administration. Binding potential (BP ND) values were determined with the equilibrium method (integral interval: 63–123 min) using cerebellum as the reference region. BP ND values were significantly decreased in several examined brain regions after administration of amphetamine (range: 19–31%), MDMA (16–25%) or 5-HTP (13–31%). Reductions in [11C]AZ10419369 binding were greater in striatum than cortical regions after administration of 5-HTP, while no prominent regional differences were found for amphetamine and MDMA. In conclusion, [11C]AZ10419369 binding is sensitive to changes in 5-HT concentration induced by amphetamine, MDMA or 5-HTP. The robust changes in BP ND, following pretreatment drugs administered at clinically relevant doses, indicate that the applied PET imaging paradigms hold promise to be successfully used in future human studies.

Abstract Background The aim of this study was to compare the binding properties of several tau positron emission tomography tracers—THK5117, THK5351, T807 (also known as AV1451; flortaucipir), and PBB3—head to head in the same human brain tissue. Methods Binding assays were performed to compare the regional distribution of 3H-THK5117 and 3H-THK5351 in postmortem tissue from three Alzheimer’s disease (AD) cases and three control subjects in frontal and temporal cortices as well as in the hippocampus. Competition binding assays between THK5351, THK5117, PBB3, and T807, as well as off-target binding of THK5117 and T807 toward monoamine oxidase B (MAO-B), were performed using binding assays in brain homogenates and autoradiography of three AD cases. Results Regional binding of 3H-THK5117 and 3H-THK5351 was similar, except in the temporal cortex, which showed higher 3H-THK5117 binding. Saturation studies demonstrated two binding sites for 3H-THK5351 (K d1 = 5.6 nM, Bmax = 76 pmol/g; K d2 = 1 nM, Bmax = 40 pmol/g). Competition studies in the hippocampus between 3H-THK5351 and unlabeled THK5351, THK5117, and T807 revealed super-high-affinity sites for all three tracers (THK5351 K i = 0.1 pM; THK5117 K i = 0.3 pM; T807 K i = 0.2 pM) and an additional high-affinity site (THK5351 K i = 16 nM; THK5117 K i = 20 nM; T807 K i = 78nM). 18F-T807, 11C-THK5351, and 11C-PBB3 autoradiography of large frozen sections from three AD brains showed similar regional binding for the three tracers, with lower binding intensity for 11C-PBB3. Unlabeled THK5351 and T807 displaced 11C-THK5351 to a similar extent and a lower extent, respectively, compared with 11C-PBB3. Competition with the MAO-B inhibitor 3H-l-deprenyl was observed for THK5117 and T807 in the hippocampus (THK5117 K i = 286 nM; T807 K i = 227 nM) and the putamen (THK5117 K i = 148 nM; T807 K i = 135 nM). 3H-THK5351 binding was displaced using autoradiography competition with unlabeled THK5351 and T807 in cortical areas by 70–80% and 60–77%, respectively, in the basal ganglia, whereas unlabeled deprenyl displaced 3H-THK5351 binding by 40% in the frontal cortex and 50% in the basal ganglia. Conclusions THK5351, THK5117, and T807 seem to target similar binding sites, but with different affinities, whereas PBB3 seems to target its own binding site. Both THK5117 and T807 demonstrated off-target binding in the hippocampus and putamen with a ten times lower binding affinity to the MAO-B inhibitor deprenyl compared with 3H-THK5351.

Abstract Background Reference tissue-based quantification of brain PET data does not typically include correction for signal originating from blood vessels, which is known to result in biased outcome measures. The bias extent depends on the amount of radioactivity in the blood vessels. In this study, we seek to revisit the well-established Logan plot and derive alternative formulations that provide estimation of distribution volume ratios (DVRs) that are corrected for the signal originating from the vasculature. Results New expressions for the Logan plot based on arterial input function and reference tissue were derived, which included explicit terms for whole blood radioactivity. The new methods were evaluated using PET data acquired using [11C]raclopride and [18F]MNI-659. The two-tissue compartment model (2TCM), with which signal originating from blood can be explicitly modeled, was used as a gold standard. DVR values obtained for [11C]raclopride using the either blood-based or reference tissue-based Logan plot were systematically underestimated compared to 2TCM, and for [18F]MNI-659, a proportionality bias was observed, i.e., the bias varied across regions. The biases disappeared when optimal blood-signal correction was used for respective tracer, although for the case of [18F]MNI-659 a small but systematic overestimation of DVR was still observed. Conclusions The new method appears to remove the bias introduced due to absence of correction for blood volume in regular graphical analysis and can be considered in clinical studies. Further studies are however required to derive a generic mapping between plasma and whole-blood radioactivity levels.

Abstract Background [11C]-l-deprenyl-D2 is a positron emission tomography (PET) radioligand for measurement of the monoamine oxidase B (MAO-B) activity in vivo brain. The estimation of the test-retest reproducibility is important for accurate interpretation of PET studies. Results We performed two [11C]-l-deprenyl-D2 scans for six healthy subjects and evaluated the test-retest variability of this radioligand. MAO-B binding was quantified by two tissue compartment model (2TCM) with three rate constants (K 1, k 2, k 3) using metabolite-corrected plasma radioactivity. The λk 3 defined as (K 1/k 2) × k 3 was also calculated. The correlation between MAO-B binding and age, and the effect of partial volume effect correction (PVEc) for the reproducibility were also estimated. %difference of k 3 was 2.6% (medial frontal cortex) to 10.3% (hippocampus), and that of λk 3 was 5.0% (thalamus) to 9.2% (cerebellum). Mean %difference of all regions were 5.3 and 7.0% in k 3 and λk 3, respectively. All regions showed below 10% variabilities except the hippocampus in k 3 (10.3%). Intraclass correlation coefficient (ICC) of k 3 was 0.78 (hippocampus) to 0.98 (medial frontal cortex), and that of λk 3 was 0.78 (hippocampus) to 0.95 (thalamus). Mean ICC were 0.94 and 0.89 in k 3 and λk 3, respectively. The highest positive correlation with age was observed in the hippocampus, as r = 0.75 in k 3 and 0.76 in λk 3. After PVEc, mean %difference were 5.6 and 7.2% in k 3 and λk 3, respectively. Mean ICC were 0.92 and 0.90 for k 3 and λk 3, respectively. These values were almost the same as those before PVEc. Conclusions The present results indicate that k 3 and λk 3 of [11C]-l-deprenyl-D2 are reliable parameters for test-retest reproducibility with healthy subjects both before and after PVEc. The studies with patients of larger sample size are required for further clinical applications.

Traumatic brain injury (TBI) modelled by lateral fluid percussion-induction (LFPI) in rats is a widely used experimental rodent model to explore and understand the underlying cellular and molecular alterations in the brain caused by TBI in humans. Current improvements in imaging with positron emission tomography (PET) have made it possible to map certain features of TBI-induced cellular and molecular changes equally in humans and animals. The PET imaging technique is an apt supplement to nanotheranostic-based treatment alternatives that are emerging to tackle TBI. The present study aims to investigate whether the two radioligands, [11C]PBR28 and [18F]flumazenil, are able to accurately quantify in vivo molecular-cellular changes in a rodent TBI-model for two different biochemical targets of the processes. In addition, it serves to observe any palpable variations associated with primary and secondary injury sites, and in the affected versus the contralateral hemispheres. As [11C]PBR28 is a radioligand of the 18 kD translocator protein, the up-regulation of which is coupled to the level of neuroinflammation in the brain, and [18F]flumazenil is a radioligand for GABAA-benzodiazepine receptors, whose level mirrors interneuronal activity and eventually cell death, the use of the two radioligands may reveal two critical features of TBI. An up-regulation in the [11C]PBR28 uptake triggered by the LFP in the injured (right) hemisphere was noted on day 14, while the uptake of [18F]flumazenil was down-regulated on day 14. When comparing the left (contralateral) and right (LFPI) hemispheres, the differences between the two in neuroinflammation were obvious. Our results demonstrate a potential way to measure the molecular alterations in a rodent-based TBI model using PET imaging with [11C]PBR28 and [18F]flumazenil. These radioligands are promising options that can be eventually used in exploring the complex in vivo pharmacokinetics and delivery mechanisms of nanoparticles in TBI treatment.

Associations between dopamine receptor levels and pro- and antisocial behavior have previously been demonstrated in human subjects using positron emission tomography (PET) and self-rated measures of personality traits. So far, only one study has focused on the dopamine D1-receptor (D1-R), finding a positive correlation with the trait social desirability, which is characterized by low dominant and high affiliative behavior, while physical aggression showed a negative correlation. The aim of the present study was to replicate these previous findings using a new independent sample of subjects.Twenty-six healthy males were examined with the radioligand [11C]SCH-23390, and completed the Swedish universities Scales of Personality (SSP) which includes measures of social desirability and physical trait aggression. The simplified reference tissue model with cerebellum as reference region was used to calculate BPND values in the whole striatum and limbic striatum. The two regions were selected since they showed strong association between D1-R availability and personality scores in the previous study. Pearson's correlation coefficients and replication Bayes factors were then employed to assess the replicability and robustness of previous results.There were no significant correlations (all p values > 0.3) between regional BPND values and personality scale scores. Replication Bayes factors showed strong to moderate evidence in favor no relationship between D1-receptor availability and social desirability (striatum BF01 = 12.4; limbic striatum BF01 = 7.2) or physical aggression scale scores (limbic striatum BF01 = 3.3), compared to the original correlations.We could not replicate the previous findings of associations between D1-R availability and either pro- or antisocial behavior as measured using the SSP. Rather, there was evidence in favor of failed replications of associations between BPND and scale scores. Potential reasons for these results are restrictive variance in both PET and personality outcomes due to high sample homogeneity, or that the previous findings were false positives.

Intra-striatal transplantation of fetal ventral mesencephalic (VM) tissue has a therapeutic effect on patients with Parkinson’s disease (PD). Sertoli cells (SCs) possess immune-modulatory properties that benefit transplantation. We hypothesized that co-graft of SCs with VM tissue can attenuate rejection. Hemi-parkinsonian rats were generated by injecting 6-hydroxydopamine into the right medial forebrain bundle of Sprague Dawley (SD) rats. The rats were then intrastriatally transplanted with VM tissue from rats or pigs (rVM or pVM), with/without a co-graft of SCs (rVM+SCs or pVM+SCs). Recovery of dopaminergic function and survival of the grafts were evaluated using the apomorphine-induced rotation test and small animal-positron emission tomography (PET) coupled with [18F] DOPA or [18F] FE-PE2I, respectively. Immunohistochemistry (IHC) examination was used to determine the survival of the grafted dopaminergic neurons in the striatum and to investigate immune-modulatory effects of SCs. The results showed that the rVM+SCs and pVM+SCs groups had significantly improved drug-induced rotational behavior compared with the VM alone groups. PET revealed a significant increase in specific uptake ratios (SURs) of [18F] DOPA and [18F] FE-PE2I in the grafted striatum of the rVM+SCs and pVM+SCs groups as compared to that of the rVM and pVM groups. SC and VM tissue co-graft led to better dopaminergic (DA) cell survival. The co-grafted groups exhibited lower populations of T-cells and activated microglia compared to the groups without SCs. Our results suggest that co-graft of SCs benefit both xeno- and allo-transplantation of VM tissue in a PD rat model. Use of SCs enhanced the survival of the grafted dopaminergic neurons and improved functional recovery. The enhancement may in part be attributable to the immune-modulatory properties of SCs. In addition, [18F]DOPA and [18F]FE-PE2I coupled with PET may provide a feasible method for in vivo evaluation of the functional integrity of the grafted DA cell in parkinsonian rats.

In quantitative PET measurements, the analysis of radiometabolites in plasma is essential for determining the exact arterial input function. Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT) and it is therefore important to investigate their radiometabolism. We have chosen to explore the radiometabolic profile of [11C]MADAM, one of these radioligands widely used for in vivo PET-SERT studies. The metabolism of [11C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the radiometabolic rate of the radioligand [11C]MADAM in vitro and in vivo was examined by radio-HPLC. RLM and HLM incubations were carried out at two different carrier concentrations of 1 and 10 μM. Urine samples after perfusion of [11C]MADAM/MADAM in rats were also analysed by radio-HPLC. Analysis by UHPLC/Q-ToF-MS identified the metabolites produced in vitro to be results of N-demethylation, S-oxidation and benzylic hydroxylation. The presence of carrier greatly affected the radiometabolism rate of [11C]MADAM in both RLM/HLM experiments and in vivo rat studies. The good concordance between the results predicted by RLM and HLM experiments and the in vivo data obtained in rat studies indicate that the kinetics of the radiometabolism of the radioligand [11C]MADAM is dose-dependent. This issue needs to be addressed when the diarylsulfide class of compounds are used in PET quantifications of SERT.

Molecular imaging of the 18 kD Translocator protein (TSPO) with positron emission tomography (PET) is of great value for studying neuroinflammation in rodents longitudinally. Quantification of the TSPO in rodents is, however, quite challenging. There is no suitable reference region and the use of plasma-derived input is not an option for longitudinal studies. The aim of this study was therefore to evaluate the use of the standardized uptake value (SUV) as an outcome measure for TSPO imaging in rodent brain PET studies, using [11C]PBR28. In the first part of the study, healthy male Wistar rats (n = 4) were used to determine the correlation between the distribution volume (VT, calculated with Logan graphical analysis) and the SUV. In the second part, healthy male Wistar rats (n = 4) and healthy male C57BL/6J mice (n = 4), were used to determine the test-retest variability of the SUV, with a 7-day interval between measurements. Dynamic PET scans of 63 minutes were acquired with a nanoScan PET/MRI and nanoScan PET/CT. An MRI scan was made for anatomical reference with each measurement. The whole brain VT of [11C]PBR28 in rats was 42.9 ± 1.7. A statistically significant correlation (r2 = 0.96; p < 0.01) was found between the VT and the SUV. The test-retest variability in 8 brain region ranged from 8 to 20% in rats and from 7 to 23% in mice. The interclass correlation coefficient (ICC) was acceptable to excellent for rats, but poor to acceptable for mice.The SUV of [11C]PBR28 showed a high correlation with VT as well as good test-retest variability. For future longitudinal small animal PET studies the SUV can thus be used to describe [11C]PBR28 uptake in healthy brain tissue. Based on the present observations, further studies are needed to explore the applicability of this approach in small animal disease models, with special regard to neuroinflammatory models.

Alzheimer's disease (AD) is a devastating disease affecting predominantly the aging population. One of the characteristic pathological hallmarks of AD are neuritic plaques, consisting of amyloid-β peptide (Aβ). While there has been some advancement in diagnostic classification of AD patients according to their clinical severity, no fully reliable method for pre-symptomatic diagnosis of AD is available. To enable such early diagnosis, which will allow the initiation of treatments early in the disease progress, neuroimaging tools are under development, making use of Aβ-binding ligands that can visualize amyloid plaques in the living brain. Here we investigate the properties of a newly designed series of D-enantiomeric peptides which are derivatives of ACI-80, formerly called D1, which was developed to specifically bind aggregated Aβ1-42. We describe ACI-80 derivatives with increased stability and Aβ binding properties, which were characterized using surface plasmon resonance and enzyme-linked immunosorbent assays. The specific interactions of the lead compounds with amyloid plaques were validated by ex vivo immunochemistry in transgenic mouse models of AD. The novel compounds showed increased binding affinity and are promising candidates for further development into in vivo imaging compounds.