The P2X7 receptor is an adenosine triphosphate–gated ion channel, which is abundantly expressed in glial cells within the central nervous system and in the periphery. P2X7 receptor activation leads to the release of the proinflammatory cytokine IL-1β in the brain, and antagonism of the P2X7 receptor is a novel therapeutic strategy to dampen adenosine triphosphate–dependent IL-1β signaling. PET ligands for the P2X7 receptor will not only be valuable to assess central target engagement of drug candidates but also hold promise as surrogate markers of central neuroinflammation. Herein we describe the in vitro and in vivo evaluation of 18F-JNJ-64413739, an 18F-labeled PET ligand for imaging the P2X7 receptor in the brain. Methods: P2X7 receptor affinity and specificity, pharmacokinetics, metabolic stability, blood–brain barrier permeability, and off-target binding of JNJ-64413739 were evaluated in a series of in vitro, ex vivo, and in vivo assays. 18F-JNJ-64413739 was radiolabeled via a one-step nucleophilic aromatic substitution. The tracer was also studied in rhesus macaques, and PET images were analyzed with an arterial plasma input function–based Logan graphical analysis. Results: The potency (half-maximal inhibitory concentration) of the P2X7 receptor antagonist JNJ-64413739 is 1.0 ± 0.2 nM and 2.0 ± 0.6 nM at the recombinant human and rat P2X7 receptor, respectively, and the binding affinity is 2.7 nM (rat cortex binding assay) and 15.9 nM (human P2X7 receptor). In nonhuman primate PET imaging studies, dose-dependent receptor occupancy of JNJ-54175446 was observed in 2 rhesus monkeys. At a 0.1 mg/kg dose (intravenous) of JNJ-54175446, the receptor occupancy was calculated to be 17% by Logan graphical analysis, whereas a dose of 2.5 mg/kg yielded a receptor occupancy of 60%. Conclusion: The preclinical evaluation of 18F-JNJ-64413739 demonstrates that the tracer engages the P2X7 receptor. Reproducible and dose-dependent receptor occupancy studies with the P2X7 receptor antagonist JNJ-54175446 were obtained in rhesus monkeys. This novel PET tracer exhibits in vitro and in vivo characteristics suitable for imaging the P2X7 receptor in the brain and warrants further studies in humans.

The P2X7 receptor, an adenosine triphosphate (ATP)-gated purinoreceptor, has emerged as one of the key players in neuroinflammatory processes. Therefore, developing a positron emission tomography (PET) tracer for imaging of P2X7 receptors in vivo presents a promising approach to diagnose, monitor, and study neuroinflammation in a variety of brain disorders. To fulfill the goal of developing a P2X7 PET ligand as a biomarker of neuroinflammation, [18F]JNJ-64413739 has been recently disclosed. We evaluated [18F]JNJ-64413739 in a rat model of neuroinflammation induced by an intracerebral injection of lipopolysaccharide (LPS). In vivo brain uptake was determined by PET imaging. Upregulation of neuroinflammatory biomarkers was determined by quantitative polymerase chain reaction (qPCR). Distribution of the tracer in the brain was determined by ex vivo autoradiography (ARG). The specificity of [18F]JNJ-64413739 was confirmed by performing blocking experiments with the P2X7 antagonist JNJ-54175446. Brain regions of rats injected with LPS had a significantly increased uptake (34 % ± 3 % s.e.m., p = 0.036, t test, standardized uptake value measured over the entire scanning period) of [18F]JNJ-64413739 relative to the corresponding brain regions of control animals injected with phosphate-buffered saline (PBS). The uptake in the contralateral regions and cerebellum was not significantly different between the groups of animals. The increase in uptake of [18F]JNJ-64413739 at the LPS-injected site observed by PET imaging was concordant with ex vivo ARG, upregulation of neuroinflammatory biomarkers, and elevated P2X7 expression levels. While further work is needed to study [18F]JNJ-64413739 in other types of neuroinflammation, the current results favorably characterize [18F]JNJ-64413739 as a potential PET tracer of central neuroinflammation.

In Alzheimer's disease, the density and spread of aggregated tau protein track well with neurodegeneration and cognitive decline, making the imaging of aggregated tau a compelling biomarker. A structure-activity relationship exploration around an isoquinoline hit, followed by an exploration of tolerated fluorination positions, allowed us to identify 9 (JNJ-64326067), a potent and selective binder to aggregated tau with a favorable pharmacokinetic profile and no apparent off-target binding. This was confirmed in rat and monkey positron emission tomography studies using [18F]9.

Aggregates of hyperphosphorylated tau (PHF-tau), such as neurofibrillary tangles, are linked to the degree of cognitive impairment in Alzheimer's disease. We have recently reported early clinical results of a novel PHF-tau targeting PET imaging agent, [F18]-T807. Since then, we have investigated a second novel PHF-tau targeting PET imaging agent, [F18]-T808, with different pharmacokinetic characteristics, which may be favorable for imaging Alzheimer's disease and other tauopathies. Here, we describe the first human brain images with [F18]-T808.

PURPOSE:We identified and validated [(18)F]-CP18, a DEVD (the caspase 3 substrate recognition motif) containing substrate-based compound as an imaging tracer for caspase-3 activity in apoptotic cells.PROCEDURES:CP18 was radiolabeled with fluorine-18 using click chemistry. The affinity and selectivity of CP18 for caspase-3 were evaluated in vitro. The biodistribution and metabolism pattern of [(18)F]-CP18 were assessed in vivo. [(18)F]-CP18 positron emission tomography (PET) scans were performed in a dexamethasone-induced thymic apoptosis mouse model. After imaging, the mice were sacrificed, and individual organs were collected, measured in a gamma counter, and tested for caspase-3 activity.RESULTS:In vitro enzymatic caspase-3 assay demonstrated specific cleavage of CP18. In vivo, [(18)F]-CP18 is predominantly cleared through the kidneys and urine, and is rapidly eliminated from the bloodstream. There was a sixfold increase in caspase activity and a fourfold increase of [(18)F]-CP18 retention in the dexamethasone-induced thymus of treated versus control mice.CONCLUSIONS:We report the use [(18)F]-CP18 as a PET tracer for imaging apoptosis. Our data support further development of this tracer for clinical PET applications.

Objective: We wished to develop a highly selective positron emission tomography (PET) imaging agent targeting PHF-tau in human Alzheimer's disease (AD) brains.Methods: To screen potential tau binders, human AD brain sections were used as a source of native paired helical filament (PHF)-tau and A beta rather than synthetic tau aggregates or A beta fibrils generated in vitro to measure the affinity and selectivity of [F-18]T807 to tau and All Brain uptake and biodistribution of [F-18]T807 in mice were also tested.Results: In vitro autoradiography results show that [F-18]T807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant (K-d) of [F-18]T807 (14.6 nM) was measured using brain sections from the frontal lobe of AD patients. A comparison of autoradiography and double immunohistochemical staining of PHF-tau and A beta on adjacent sections demonstrated that [F-18]T807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight A beta plaques. In vivo studies in mice demonstrated that [F-18]T807 was able to cross the blood brain barrier and washed out quickly.Conclusions: [F-18]T807 demonstrates high affinity and selectivity to PHF-tau as well as favorable in vivo properties, making this a promising candidate as an imaging agent for AD. (C) 2013 The Alzheimer's Association. All rights reserved.

PURPOSE:A novel caspase-3 substrate-based probe [(18)F]-CP18 was evaluated as an in vivo positron emission tomography (PET) imaging agent for monitoring apoptosis in tumors.METHODS:Uptake of [(18)F]-CP18 in cell assays and tumors was measured. Caspase-3/7 activities in cell lysates and tumor homogenates were determined. Autoradiography,Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and cleaved caspase-3 immunostaining were performed on adjacent tumor sections to identify areas of apoptosis.RESULTS:The in vitro cell assays showed caspase-3-dependent uptake of [(18)F]-CP18 in tumor cells when treated with an apoptosis inducer. The in vivo microPET imaging signal of [(18)F]-CP18 in xenograft tumors correlated with the ex vivo caspase-3/7 activities in these tumors. Furthermore, tumor autoradiographies of [(18)F]-CP18 in tumor sections matched adjacent sections stained by TUNEL and caspase-3 immunohistochemistry (IHC).CONCLUSIONS:[(18)F]-CP18 demonstrated high affinity and selectivity for activated caspase-3 both in vitro and in vivo, and the results support [(18)F]-CP18 as a promising new PET imaging agent for apoptosis.

Senile plaques and neurofibrillary tangles are prominent neuropathological hallmarks in Alzheimer's disease and are considered to be targets for therapeutic intervention as well as biomarkers for diagnostic in vivo imaging agents. While there are a number of amyloid-β positron emission tomography (PET) tracers currently in different stages of clinical development and commercialization, there have been very few reports on imaging agents selectively targeting tau aggregates. In search of [18F]-PET tracers that possess great binding affinity and selectivity toward tau tangles, we tested more than 900 compounds utilizing a unique screening process. A competitive autoradiography assay was set up to test compounds for binding to native tau tangles and amyloid-β plaques on human brain tissue sections. In our in vitro assays, the 18F labeled compound [18F]-T808 displayed a high level of binding affinity and good selectivity for tau aggregates over amyloid-β plaques. [18F]-T808 showed rapid uptake and washout in rodent brains. Our in vitro and preclinical in vivo studies suggest that [18F]-T808 possesses suitable properties and characteristics to be a specific and selective PET probe for imaging of paired helical filament tau in human brains.

Tissue kallikrein (KLK1) processes low-molecular weight kininogen to produce vasoactive kinins, which exert biological functions via kinin receptor signaling. Using various delivery approaches, we have demonstrated that tissue kallikrein through kinin B2 receptor signaling exhibits a wide spectrum of beneficial effects by reducing cardiac and renal injuries, restenosis and ischemic stroke, and by promoting angiogenesis and skin wound healing, independent of blood pressure reduction. Protection by tissue kallikrein in oxidative organ damage is attributed to the inhibition of apoptosis, inflammation, hypertrophy and fibrosis. Tissue kallikrein also enhances neovascularization in ischemic heart and limb. Moreover, tissue kallikrein/kinin infusion not only prevents but also reverses kidney injury, inflammation and fibrosis in salt-induced hypertensive rats. Furthermore, there is a wide time window for kallikrein administration in protection against ischemic brain infarction, as delayed kallikrein infusion for 24 h after cerebral ischemia in rats is effective in reducing neurological deficits, infarct size, apoptosis and inflammation. Importantly, in the clinical setting, human tissue kallikrein has been proven to be effective in the treatment of patients with acute brain infarction when injected within 48 h after stroke onset. Finally, kallikrein promotes skin wound healing and keratinocyte migration by direct activation of protease-activated receptor 1.