Esther J.M. Kooijman

Synthesis and initial preclinical evaluation of the P2X7 receptor antagonist [11C]A-740003 as a novel tracer of neuroinflammation

Neuroinflammation, in particular activation of microglia, is thought to play an important role in the progression of neurodegenerative diseases. In activated microglia, the purinergic P2X7 receptor is upregulated. A-740003, a highly affine and selective P2X7 receptor antagonist, is a promising candidate for the development of a radiotracer for imaging of neuroinflammation by positron emission tomography. For this purpose, [(11)C]A-740003 was synthesised and evaluated in vivo with respect to both tracer metabolism and biodistribution. In plasma, a moderate metabolic rate was seen. In healthy rat brain, only marginal uptake of [(11)C]A-740003 was observed and, therefore, metabolites in brain could not be determined. Whether the minimal brain uptake is due to the low expression levels of the P2X7 receptor in healthy brain or to limited transport across the blood-brain barrier has yet to be elucidated.

Development of carbon-11 labeled acryl amides for selective PET imaging of active tissue transglutaminase

INTRODUCTION Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme capable of forming metabolically and mechanically stable crosslinks between the γ-carboxamide of a glutamine acyl-acceptor substrate and the ε-amino functionality of a lysine acyl-donor substrate resulting in protein oligomers. High TG2 crosslinking activity has been implicated in the pathogenesis of various diseases including celiac disease, cancer and fibrotic and neurodegenerative diseases. Development of a PET tracer specific for active TG2 provides a novel tool to further investigate TG2 biology in vivo in disease states. Recently, potent irreversible active site TG2 inhibitors carrying an acrylamide warhead were synthesized and pharmacologically characterized. METHODS Three of these inhibitors, compound 1, 2 and 3, were successfully radiolabeled with carbon-11 on the acrylamide carbonyl position using a palladium mediated [(11)C]CO aminocarbonylation reaction. Ex vivo biodistribution and plasma stability were evaluated in healthy Wistar rats. Autoradiography was performed on MDA-MB-231 tumor sections. RESULTS [(11)C]1, -2 and -3 were obtained in decay corrected radiochemical yields of 38-55%. Biodistribution showed low uptake in peripheral tissues, with the exception of liver and kidney. Low brain uptake of <0.05% ID/g was observed. Blood plasma analysis demonstrated that [(11)C]1 and [(11)C]2 were rapidly metabolized, whereas [(11)C]3 was metabolized at a more moderate rate (63.2 ± 6.8 and 28.7 ± 10.8% intact tracer after 15 and 45 min, respectively). Autoradiography with [(11)C]3 on MDA-MB-231 tumor sections showed selective and specific binding of the radiotracer to the active state of TG2. CONCLUSIONS Taken together, these results identify [(11)C]3 as the most promising of the three compounds tested for development as PET radiotracer for the in vivo investigation of TG2 activity.

Preclinical evaluation of [18F]PK-209, a new PET ligand for imaging the ion-channel site of NMDA receptors

INTRODUCTION The present study was designed to assess whether [(18)F]PK-209 (3-(2-chloro-5-(methylthio)phenyl)-1-(3-([(18)F]fluoromethoxy)phenyl)-1-methylguanidine) is a suitable ligand for imaging the ion-channel site of N-methyl-D-aspartate receptors (NMDArs) using positron emission tomography (PET). METHODS Dynamic PET scans were acquired from male rhesus monkeys over 120min, at baseline and after the acute administration of dizocilpine (MK-801, 0.3mg/kg; n=3/condition). Continuous and discrete arterial blood samples were manually obtained, to generate metabolite-corrected input functions. Parametric volume-of-distribution (VT) images were obtained using Logan analysis. The selectivity profile of PK-209 was assessed in vitro, on a broad screen of 79 targets. RESULTS PK-209 was at least 50-fold more selective for NMDArs over all other targets examined. At baseline, prolonged retention of radioactivity was observed in NMDAr-rich cortical regions relative to the cerebellum. Pretreatment with MK-801 reduced the VT of [(18)F]PK-209 compared with baseline in two of three subjects. The rate of radioligand metabolism was high, both at baseline and after MK-801 administration. CONCLUSIONS PK-209 targets the intrachannel site with high selectivity. Imaging of the NMDAr is feasible with [(18)F]PK-209, despite its fast metabolism. Further in vivo evaluation in humans is warranted.

Synthesis and preclinical evaluation of carbon-11 labelled N-((5-(4-fluoro-2-[11C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine as a PET tracer for NR2B subunit-containing NMDA receptors

INTRODUCTION The N-methyl-D-Aspartate (NMDA) receptor plays an important role in learning and memory. Overactivation is thought to play an important role in neurodegenerative disorders such as Alzheimers disease. Currently, it is not possible to assess N-methyl-D-aspartate receptor (NMDAr) bio-availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NR2B binding site of the NMDA receptor. METHODS N-((5-(4-fluoro-2-methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was radiolabelled with carbon-11 in the phenyl moiety. Biodistribution and blocking studies were carried out in anaesthetized mice and in non-anaesthetized rats. RESULTS N-((5-(4-fluoro-2-[(11)C]methoxyphenyl)pyridin-3-yl)methyl)cyclopentanamine was prepared in 49±3% (decay-corrected) yield, affording 4.1±0.3 GBq of formulated product at the end of synthesis with a radiochemical purity of >99% and with a specific activity of 78±10 GBq/μmol. CONCLUSION A new NR2B PET ligand was developed in high yield. [(11)C]4 readily enters the brain and binds to the NR2B subunit-containing NMDAr in the rodent brain. High sigma-1 receptor binding may, however, limit its future application as a PET probe for imaging the NR2B subunit-containing NMDAr. Anaesthesia has an effect on NMDAr function and therefore can complicate interpretation of preclinical in vivo results. In addition, effects of endogenous compounds cannot be excluded. Despite these potential limitations, further studies are warranted to investigate the values of [(11)C]4 as an NR2B PET ligand.

Subchronic treatment with phencyclidine in adolescence leads to impaired exploratory behavior in adult rats without altering social interaction or N-methyl-D-aspartate receptor binding levels

Although both the onset of schizophrenia and human phencyclidine (PCP) abuse typically present within the interval from adolescence to early adulthood, the majority of preclinical research employing the PCP model of schizophrenia has been conducted on neonatal or adult animals. The present study was designed to evaluate the behavioral and neurochemical sequelae of subchronic exposure to PCP in adolescence. Male 35–42‐day‐old Sprague Dawley rats were subcutaneously administered either saline (10 ml · kg−1) or PCP hydrochloride (10 mg · kg−1) once daily for a period of 14 days (n = 6/group). The animals were allowed to withdraw from treatment for 2 weeks, and their social and exploratory behaviors were subsequently assessed in adulthood by using the social interaction test. To examine the effects of adolescent PCP administration on the regulation of N‐methyl‐D‐aspartate receptors (NMDARs), quantitative autoradiography was performed on brain sections of adult, control and PCP‐withdrawn rats by using 20 nM 3H‐MK‐801. Prior subchronic exposure to PCP in adolescence had no enduring effects on the reciprocal contact and noncontact social behavior of adult rats. Spontaneous rearing in response to the novel testing arena and time spent investigating its walls and floor were reduced in PCP‐withdrawn animals compared with control. The long‐term behavioral effects of PCP occurred in the absence of persistent deficits in spontaneous locomotion or self‐grooming activity and were not mediated by altered NMDAR density. Our results document differential effects of adolescent PCP administration on the social and exploratory behaviors of adult rats, suggesting that distinct neurobiological mechanisms are involved in mediating these behaviors.

Development of fluorine-18 labeled peptidic PET tracers for imaging active tissue transglutaminase

INTRODUCTION The protein-protein crosslinking activity of the enzyme tissue transglutaminase (TG2; EC 2.3.2.13) is associated with the pathogenesis of various diseases, including celiac disease, lung-, liver- and kidney fibrosis, cancer and neurodegenerative diseases. This study aims at developing a TG2 PET tracer based on the peptidic irreversible TG2 inhibitor Z006. METHODS Initially, the carbon-11 labeling of Z006 at the diazoketone position was explored. Subsequently, a set of analogues that allow for fluorine-18 labeling was synthesized. Two potent analogues, 6f and 6g, were radiolabeled with fluorine-18 and biodistribution and metabolite analysis in Wistar rats was performed. The identity of the main metabolite of [18F]6g was elucidated using LC-MS/MS. In vitro binding to isolated TG2 and in vitro autoradiography on MDA-MB-231 breast cancer tissue using [18F]6g was performed. RESULTS [18F]6f and [18F]6g were obtained in 20 and 9% yields, respectively. Following administration to healthy Wistar rats, rapid metabolism of both tracers was observed. Remarkably, full conversion to just one single metabolite was observed for one of the tracers, [18F]6g. By LC-MS/MS analysis this metabolite was identified as C-terminally saponified [18F]6g. This metabolite was also found to be a potent TG2 inhibitor in vitro. In vitro binding to isolated TG2 and in vitro autoradiography on MDA-MB-231 tumor sections using [18F]6g demonstrated high specific and selective binding of [18F]6g to active TG2. CONCLUSIONS Whereas based on the intensive metabolism [18F]6f seems unsuitable as a TG2 PET tracer, the results warrant further evaluation of [18F]6gin vivo.

Synthesis and Evaluation of New Fluorine-18 Labeled Verapamil Analogs To Investigate the Function of P-Glycoprotein in the Blood–Brain Barrier

P-glycoprotein is an efflux transporter located in the blood–brain barrier. (R)-[11C]Verapamil is widely used as a PET tracer to investigate its function in patients with epilepsy, Alzheimer’s disease, and other neurodegenerative diseases. Currently it is not possible to use this successful tracer in clinics without a cyclotron, because of the short half-life of carbon-11. We developed two new fluorine-18 labeled (R)-verapamil analogs, with the benefit of a longer half-life. The synthesis of (R)-N-[18F]fluoroethylverapamil ([18F]1) and (R)-O-[18F]fluoroethylnorverapamil ([18F]2) has been described. [18F]1 was obtained in reaction of (R)-norverapamil with the volatile [18F]fluoroethyltriflate acquired from bromoethyltosylate and a silver trilate column with a radiochemical yield of 2.7% ± 1.2%. [18F]2 was radiolabeled by direct fluorination of precursor 13 and required final Boc-deprotection with TFA resulting in a radiochemical yield of 17.2% ± 9.9%. Both tracers, [18F]1 and [18F]2, were administered to Wistar rats, and blood plasma and brain samples were analyzed for metabolic stability. Using [18F]1 and [18F]2, PET scans were performed in Wistar rats at baseline and after blocking with tariquidar, showing a 3.6- and 2.4-fold increase in brain uptake in the blocked rats, respectively. In addition, for both [18F]1 and [18F]2, PET scans in Mdr1a/b(−/−), Bcrp1(−/−), and WT mice were acquired, in which [18F]2 showed a more specific brain uptake in Mdr1a/b(−/−) mice and no increased signal in Bcrp1(−/−) mice. [18F]2 was selected as the best performing tracer and should be evaluated further in clinical studies.

Identification of the allosteric P2X7 receptor antagonist [11C]SMW139 as a PET tracer of microglial activation

The P2X7 receptor plays a significant role in microglial activation, and as a potential drug target, the P2X7 receptor is also an interesting target in positron emission tomography. The current study aimed at the development and evaluation of a potent tracer targeting the P2X7 receptor, to which end four adamantanyl benzamide analogues with high affinity for the human P2X7 receptor were labelled with carbon-11. All four analogues could be obtained in excellent radiochemical yield and high radiochemical purity and molar activity, and all analogues entered the rat brain. [11C]SMW139 showed the highest metabolic stability in rat plasma, and showed high binding to the hP2X7 receptor in vivo in a hP2X7 receptor overexpressing rat model. Although no significant difference in binding of [11C]SMW139 was observed between post mortem brain tissue of Alzheimer’s disease patients and that of healthy controls in in vitro autoradiography experiments, [11C]SMW139 could be a promising tracer for P2X7 receptor imaging using positron emission tomography, due to high receptor binding in vivo in the hP2X7 receptor overexpressing rat model. However, further investigation of both P2X7 receptor expression and binding of [11C]SMW139 in other neurological diseases involving microglial activation is warranted.

Synthesis and Preclinical Evaluation of the First Carbon-11 Labeled PET Tracers Targeting Substance P1-7

Two potent SP1–7 peptidomimetics have been successfully radiolabeled via [11C]CO2-fixation with excellent yields, purity, and molar activity. l-[11C]SP1–7-peptidomimetic exhibited promising ex vivo biodistribution profile. Metabolite analysis showed that l-[11C]SP1–7-peptidomimetic is stable in brain and spinal cord, whereas rapid metabolic degradation occurs in rat plasma. Metabolic stability can be significantly improved by substituting l-Phe for d-Phe, preserving 70% more of intact tracer and resulting in better brain and spinal cord tracer retention. Positron emission tomography (PET) scanning confirmed moderate brain (1.5 SUV; peak at 3 min) and spinal cord (1.0 SUV; peak at 10 min) uptake for l- and d-[11C]SP1–7-peptidomimetic. A slight decrease in SUV value was observed after pretreatment with natural peptide SP1–7 in spinal cord for l-[11C]SP1–7-peptidomimetic. On the contrary, blocking using cold analogues of l- and d-[11C]tracers did not reduce the tracers’ brain and spinal cord exposure. In summary, PET scanning of l- and d-[11C]SP1–7-peptidomimetics confirms rapid blood–brain barrier and blood–spinal-cord barrier penetration. Therefore, further validation of these two tracers targeting SP1–7 is needed in order to define a new PET imaging target and select its most appropriate radiopharmaceutical.