Sofie Celen

Preclinical Evaluation of 18F-JNJ41510417 as a Radioligand for PET Imaging of Phosphodiesterase-10A in the Brain

Phosphodiesterases are enzymes that inactivate the intracellular second messengers 3′,5′-cyclic adenosine-monophosphate and/or cyclic guanosine-monophosphate. Of all 11 known phosphodiesterase families, phosphodiesterase-10A (PDE10A) has the most restricted distribution, with high expression in the striatum. PDE10A inhibitors are pursued as drugs for treatment of neuropsychiatric disorders. We have synthesized and evaluated 18F-JNJ41510417 as a selective and high-affinity radioligand for in vivo brain imaging of PDE10A using PET. Methods: The biodistribution of 18F-JNJ41510417 was evaluated in rats. Rat plasma and perfused brain homogenates were analyzed by high-performance liquid chromatography to quantify radiometabolites. Dynamic small-animal PET was performed in rats and in wild-type and PDE10A knock-out mice and compared with ex vivo autoradiography. Blocking and displacement experiments were performed using the nonradioactive analog and other selective PDE10A inhibitors. Results: Tissue distribution studies showed predominant hepatobiliary excretion, sufficient brain uptake (0.56 ± 0.00 percentage injected dose at 2 min after tracer injection), and continuous accumulation of the tracer in the striatum over time; rapid washout of nonspecific binding from other brain regions was observed. Polar radiometabolites were detected in plasma and brain tissue. Dynamic small-animal PET showed continuous tracer accumulation in the striatum, with rapid decline in the cortex and cerebellum. Pretreatment and chase experiments with PDE10A inhibitors showed that the tracer binding to PDE10A was specific and reversible. Imaging in PDE10A knock-out and wild-type mice further confirmed that binding in the striatum was specific for PDE10A. Conclusion: Experiments in rats and PDE10A knock-out mice indicate that 18F-JNJ41510417 binds specifically and reversibly to PDE10A in the striatum, suggesting that this new fluorinated quinoline derivative is a promising candidate for in vivo imaging of PDE10A using PET.

Synthesis, In Vivo Occupancy, and Radiolabeling of Potent Phosphodiesterase Subtype-10 Inhibitors as Candidates for Positron Emission Tomography Imaging

We have recently reported the phosphodiesterase 10A (PDE10A) inhibitor 2-[4-[1-(2-[(18)F]fluoroethyl)-4-pyridin-4-yl-1H-pyrazol-3-yl]-phenoxymethyl]-quinoline ([(18)F]1a) as a promising candidate for in vivo imaging using positron emission tomography (PET). We now describe the synthesis and biological evaluation of a series of related pyridinyl analogues that exhibit high potency and selectivity as PDE10A inhibitors. The most interesting compounds were injected in rats to measure their levels of PDE10A occupancy through an in vivo occupancy assay. The 3,5-dimethylpyridine derivative 3 and the 5-methoxypyridine derivative 4 showed a comparable level of occupancy to that of 1a. Because these derivatives showed lower in vitro activity and are slightly less lipophilic than 1a, we hypothesized that they could behave as better PET imaging ligands. Compounds [(18)F]3, [(18)F]4, and [(11)C]4 were radiosynthesized and subjected to biodistribution studies in rats for a preliminary evaluation as candidate PET radioligands for in vivo imaging of PDE10A in the brain.

Quantification of 18F-JNJ-42259152, a Novel Phosphodiesterase 10A PET Tracer: Kinetic Modeling and Test–Retest Study in Human Brain

Phosphodiesterase 10A (PDE10A) plays a central role in striatal signaling and is implicated in several neuropsychiatric disorders, such as movement disorders and schizophrenia. We performed initial brain kinetic modeling of the novel PDE10A tracer 18F-JNJ-42259152 (2-[[4-[1-(2-18F-fluoroethyl)-4-(4-pyridinyl)-1H-pyrazol-3-yl]phenoxy]methyl]-3,5-dimethyl-pyridine) and studied test–retest reproducibility in healthy volunteers. Methods: Twelve healthy volunteers (5 men, 7 women; age range, 42–77 y) were scanned dynamically up to 135 min after bolus injection of 172.5 ± 10.3 MBq of 18F-JNJ42259152. Four volunteers (2 men, 2 women) underwent retest scanning, with a mean interscan interval of 37 d. Input functions and tracer parent fractions were determined using arterial sampling and high-performance liquid chromatography analysis. Volumes of interest for the putamen, caudate nucleus, ventral striatum, substantia nigra, thalamus, frontal cortex, and cerebellum were delineated using individual volumetric T1 MR imaging scans. One-tissue (1T) and 2-tissue (2T) models were evaluated to calculate total distribution volume (VT). Simplified models were also tested to calculate binding potential (BPND), including the simplified reference tissue model (SRTM) and multilinear reference tissue model, using the frontal cortex as the optimal reference tissue. The stability of VT and BPND was assessed down to a 60-min scan time. Results: The average intact tracer half-life in blood was 90 min. The 2T model VT values for the putamen, caudate nucleus, ventral striatum, substantia nigra, thalamus, frontal cortex, and cerebellum were 1.54 ± 0.37, 0.90 ± 0.24, 0.64 ± 0.18, 0.42 ± 0.09, 0.35 ± 0.09, 0.30 ± 0.07, and 0.36 ± 0.12, respectively. The 1T model provided significantly lower VT values, which were well correlated to the 2T VT. SRTM BPND values referenced to the frontal cortex were 3.45 ± 0.43, 1.78 ± 0.35, 1.10 ± 0.31, and 0.44 ± 0.09 for the respective target regions putamen, caudate nucleus, ventral striatum, and substantia nigra, with similar values for the multilinear reference tissue model. Good correlations were found for the target regions putamen, caudate nucleus, ventral striatum, and substantia nigra between the 2T-compartment model BPND and the SRTM BPND (r = 0.57, 0.82, 0.70, and 0.64, respectively). SRTM BPND using a 90- and 60-min acquisition interval showed low bias. Test–retest variability was 5%–19% for 2T VT and 5%–12% for BPND SRTM. Conclusion: Kinetic modeling of 18F-JNJ-42259152 shows that PDE10A activity can be reliably quantified and simplified using a reference tissue model with the frontal cortex as reference and a 60-min acquisition period.

Synthesis, Evaluation, and Radiolabeling of New Potent Positive Allosteric Modulators of the Metabotropic Glutamate Receptor 2 as Potential Tracers for Positron Emission Tomography Imaging

The synthesis and in vitro and in vivo evaluation of a new series of 7-(phenylpiperidinyl)-1,2,4-triazolo[4,3-a]pyridines, which were conveniently radiolabeled with carbon-11, as potential positron emission tomography (PET) radiotracers for in vivo imaging of the allosteric binding site of the metabotropic glutamate (mGlu) receptor subtype 2 are described. The synthesized compounds proved to be potent and selective positive allosteric modulators (PAMs) of the mGlu receptor 2 (mGluR2) in a [³⁵S]GTPγS binding assay and were able to displace an mGluR2 PAM radioligand, which we had previously developed, with IC₅₀ values in the low nanomolar range. The most promising candidates were radiolabeled and subjected to biodistribution studies and radiometabolite analysis in rats. Preliminary small-animal PET (μPET) studies in rats indicated that [¹¹C]20f binds specifically and reversibly to an mGluR2 allosteric site, strongly suggesting that it is a promising candidate for PET imaging of mGluR2 in the brain.

Preclinical evaluation of [(18)F]JNJ42259152 as a PET tracer for PDE10A.

Phosphodiesterase-10A (PDE10A) is implicated in several neuropsychiatric disorders involving basal ganglia neurotransmission, such as schizophrenia, obsessive-compulsive disorder and Huntingtons disease. To confirm target engagement and exposure-occupancy relationships of clinical candidates for treatment, and to further explore the in vivo biology of PDE10A, non-invasive imaging using a specific PET ligand is warranted. Recently we have reported the in vivo evaluation of [(18)F]JNJ41510417 which showed specific binding to PDE10A in rat striatum, but with relatively slow kinetics. A chemically related derivative JNJ42259152 was found to have a similar in vivo occupancy, but lower lipophilicity and lower PDE10A in vitro inhibitory activity compared to JNJ41510417. (18)F-labeled JNJ42259152 was therefore evaluated as a potential PDE10A PET radiotracer. Baseline PET in rats and monkey showed specific retention in the PDE10A-rich striatum, and fast wash-out, with a good contrast to non-specific binding, in other brain regions. Pretreatment and chase experiments in rats with the selective PDE10A inhibitor MP-10 showed that tracer binding was specific and reversible. Absence of specific binding in PDE10A knock-out (KO) mice further confirmed PDE10A specificity. In vivo radiometabolite analysis using high performance liquid chromatography (HPLC) showed presence of polar radiometabolites in rat plasma and brain. In vivo imaging in rat and monkey further showed faster brain kinetics, and higher striatum-to-cerebellum ratios for [(18)F]JNJ42259152 compared to [(18)F]JNJ41510417. The arterial input function corrected for radiometabolites was determined in rats and basic kinetic modeling was established. For a 60-min acquisition time interval, striatal binding potential of the intact tracer referenced to the cerebellum showed good correlation with corresponding binding potential values of a Simplified Reference Tissue Model and referenced Logan Plot, the latter using a population averaged reference tissue-to-plasma clearance rate and offering the possibility to generate representative parametric binding potential images. In conclusion we can state that in vivo imaging in PDE10A KO mice, rats and monkey demonstrates that [(18)F]JNJ42259152 provides a PDE10A-specific signal in the striatum with good pharmacokinetic properties. Although presence of a polar radiometabolite in rat brain yielded a systematic but reproducible underestimation of the striatal BPND, a Logan reference tissue model approach using 60 min acquisition data is appropriate for quantification.

Preclinical Evaluation of a P2X7 Receptor–Selective Radiotracer: PET Studies in a Rat Model with Local Overexpression of the Human P2X7 Receptor and in Nonhuman Primates

The P2X7 receptor (P2X7R) orchestrates neuroinflammation, and this is the basis for an increased interest in the development of antagonists inhibiting P2X7R function in the brain. This study provides the preclinical evaluation of 11C-JNJ-54173717, a PET tracer for P2X7R in both rats and nonhuman primates. Methods: 11C-JNJ-54173717 is a high-affinity radiotracer for the human P2X7R (hP2X7R). Biodistribution and radiometabolite studies were performed. Viral vectors encoding either enhanced green fluorescent protein-hP2X7R or 3flag-hP2X7R were engineered and validated in cell culture. hP2X7R was regionally overexpressed in the rat striatum after stereotactic injection of viral vectors. Dynamic small-animal PET studies were performed in vector-injected rats and in healthy monkeys using 11C-JNJ-54173717. Results: The affinity of JNJ-54173717 was 1.6 ± 0.1 nM in a rat cortex P2X7R membrane binding assay. In a functional assay at the recombinant human and rat P2X7R orthologs, the half maximal inhibitory concentration (IC50) of JNJ-54173717 was 4.2 ± 0.01 nM and 7.6 ± 0.01 nM, respectively. The rat biodistribution study showed that 11C-JNJ-54173717 crossed the blood–brain barrier and was cleared from plasma mainly via the hepatobiliary pathway. A polar radiometabolite was found in rat plasma. No radiometabolites were detected in rat brain. Dynamic small-animal PET showed binding of 11C-JNJ-54173717 in the striatum expressing hP2X7R, with rapid washout from the noninjected control striatum and other brain regions. Likewise, 11C-JNJ-54173717 PET signal was blocked by a chemically distinct P2X7R ligand, indicating specific binding to P2X7R in the monkey brain. Conclusion: JNJ-54173717 is a high-affinity P2X7R antagonist. An animal rat model stably expressing hP2X7R was developed and validated, identifying favorable characteristics for 11C-JNJ-54173717 as a PET radioligand for in vivo visualization of hP2X7R. 11C-JNJ-54173717 selectively visualized P2X7R in the monkey brain, and this radioligand will be further evaluated in a clinical setting to study P2X7R expression levels in neurodegenerative disorders.

Development and biological evaluation of 99mTc-sulfonamide derivatives for in vivo visualization of CA IX as surrogate tumor hypoxia markers

In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. In this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, K(i) = 33 nM for CA IX) has been conjugated with bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG2) tetradendate ligands and the conjugates radiolabelled with (99m)Tc, to obtain anionic and neutral (99m)Tc-labeled sulfonamide derivatives, respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory activities against hCA IX (K(i) = 59-66 nM). In addition, a second generation bis AEBS was conjugated with MAG2 and labeled with (99m)Tc, and the obtained diastereomers were also evaluated in targeting CA IX. Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG₂ tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28% intact). This preliminary data suggest that negatively charged (99m)Tc-labeled sulfonamide derivatives with modest lipophilicity and longer circulation time could be promising markers to target CA IX.

PET Radioligands for In Vivo Visualization of Neuroinflammation

Neuroinflammation is a well-orchestrated, dynamic, multicellular process playing a major role in neurodegenerative disorders. The microglia which make up the innate immune system of the central nervous system are key cellular mediators of neuroinflammatory processes. In normal condition they exert a protective function, providing tissue repair by releasing anti-inflammatory cytokines and neurotrophic factors. Upon neuronal injury or infection, they become overactivated, thereby releasing neurotoxic substances, amplifying neuroinflammation leading to neurodegeneration. Positron emission tomography (PET) provides a sensitive non-invasive imaging technique to study and quantify receptor and enzyme expression. A radiolabeled tracer for a protein (over)expressed in neuroinflammation and more specifically for the overactivated microglia would be useful as a diagnostic tool in the follow-up of neuroinflammation progression and to study the efficacy of anti-inflammatory therapy over time. In this manuscript, an overview of potential PET tracer targets upregulated during neuroinflammation is provided together with the current radiotracers used to image these targets. In addition, lead structures to develop radiotracers for new targets are suggested.

PET imaging of TSPO in a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide

OBJECTIVE The goal of this study was to measure functional and structural aspects of local neuroinflammation induced by intracerebral injection of lipopolysaccharide (LPS) in rats using TSPO microPET imaging with [(18)F]DPA-714, magnetic resonance imaging (MRI), in vitro autoradiography and immunohistochemistry (IHC) in order to characterize a small animal model for screening of new PET tracers targeting neuroinflammation. METHODS Rats were injected stereotactically with LPS (50 μg) in the right striatum and with saline in the left striatum. [(18)F]DPA-714 microPET, MRI, in vitro autoradiography and IHC studies were performed at different time points after LPS injection for 1 month. RESULTS Analysis of the microPET data demonstrated high uptake of the tracer in the LPS injected site with an affected-to-non-affected side-binding potential ratio (BPright-to-left) of 3.0 at 3 days after LPS injection. This BP ratio decreased gradually over time to 0.9 at 30 days after LPS injection. In vitro autoradiography ([(18)F]DPA-714) and IHC (CD68, GFAP and TSPO) confirmed local neuroinflammation in this model. Dynamic contrast enhanced (DCE) MRI demonstrated BBB breakdown near the LPS injection site at day 1, which gradually resolved over time and was absent at 1 month after LPS injection. CONCLUSION The LPS model is useful for first screening of newly developed tracers because of the easy design and the robust, unilateral inflammatory reaction allowing the use of the contralateral region as control. Additionally, this model can be used to test and follow up the benefits of anti-inflammatory therapies by non-invasive imaging.

Comparison of New Tau PET-Tracer Candidates With [18F]T808 and [18F]T807

Early clinical results of two tau tracers, [18F]T808 and [18F]T807, have recently been reported. In the present study, the biodistribution, radiometabolite quantification, and competition-binding studies were performed in order to acquire comparative preclinical data as well as to establish the value of T808 and T807 as benchmark compounds for assessment of binding affinities of eight new/other tau tracers. Biodistribution studies in mice showed high brain uptake and fast washout. In vivo radiometabolite analysis using high-performance liquid chromatography showed the presence of polar radiometabolites in plasma and brain. No specific binding of [18F]T808 was found in transgenic mice expressing mutant human P301L tau. In semiquantitative autoradiography studies on human Alzheimer disease slices, we observed more than 50% tau selective blocking of [18F]T808 in the presence of 1 µmol/L of the novel ligands. This study provides a straightforward comparison of the binding affinity and selectivity for tau of the reported radiolabeled tracers BF-158, BF-170, THK5105, lansoprazole, astemizole, and novel tau positron emission tomography ligands against T807 and T808. Therefore, these data are helpful to identify structural requirements for selective interaction with tau and to compare the performance of new highly selective and specific radiolabeled tau tracers.