Abstract 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 Aβ rather than synthetic tau aggregates or Aβ fibrils generated in vitro to measure the affinity and selectivity of 18 FT807 to tau and Aβ. Brain uptake and biodistribution of 18 FT807 in mice were also tested. Results In vitro autoradiography results show that 18 FT807 exhibits strong binding to PHF-tau-positive human brain sections. A dissociation constant ( Kd ) of 18 FT807 (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 Aβ on adjacent sections demonstrated that 18 FT807 binding colocalized with immunoreactive PHF-tau pathology, but did not highlight Aβ plaques. In vivo studies in mice demonstrated that 18 FT807 was able to cross the blood–brain barrier and washed out quickly. Conclusions 18 FT807 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.
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.
Aggregates of hyperphosphorylated tau (PHF-tau), such as neurofibrillary tangles, are linked to the degree of cognitive impairment in Alzheimer's disease. We have developed a novel PHF-tau targeting ...positron emission tomography imaging agent, F-18-T807, which may be useful for imaging Alzheimer's disease and other tauopathies. Here in, we describe the first human brain images with F-18-T807.
The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for ...identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.
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.
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.
A cell permeable, selective DPP II (also known as DPP2, DPP7, and QPP) inhibitor is reported.
A cell permeable DPP II also known as DPP2, DPP7, and quiescent cell proline dipeptidase (QPP) inhibitor ...has been synthesized. The azabicyclo3.3.0octane-based inhibitor is potent and selective and elicits very similar quiescent lymphocyte death to previously characterized inhibitors that are not as selective.
Abstract only Background: Rupture of unstable coronary artery plaque results in luminal thrombosis and myocardial infarction. Smooth muscle migration, neovascularization and apoptosis are believed to ...constitute key steps that precede plaque rupture. Identification of plaque with enhanced apoptosis may therefore allow differentation of stable from high-risk plaque. We have developed 18 FCP18, a PET tracer that is cleaved by caspase 3, a terminal enzyme in the apoptosis cascade. We sought to test the ability of this tracer to detect plaque apoptosis in an ApoE double knockout mouse model of atherosclerosis. Methods: ApoE knockout mice (n=6) were fed a high fat western diet for 30 weeks at the end of which 250µCi of 18 F-CP18 was injected intravenously. Mice were sacrificed 60 min post-injection and the aorta was excised. Ex-vivo autoradiography was performed using either whole aorta or 10 µm-thick transverse sections of the aorta mounted on glass slides. Apoptotic activity and plaque inflammation in tissue sections from similar locations in the aorta were visually assessed using TUNEL staining and CD68 antibody staining respectively. Lipid deposition was visualized by oil red O staining with an H&E counterstain. Results: In the ApoE KO mice fed a high fat diet, atherosclerotic plaque visualized by Oil Red O was present in the aortic root and aortic arch. Qualitative analysis of ex-vivo autoradiography of the aorta showed preferential uptake of 18 F-CP18 by aortic plaque at these same locations. Tunel staining and CD68 staining co-localized with plaques that exhibited increased 18 F-CP18 uptake. In contrast, in ApoE mice fed a regular diet and in wild-type mice fed the high-fat diet, there were fewer plaques in the aorta, with minimal tracer uptake and negligible histochemical evidence of macrophage influx and apoptotic activity. Conclusion: We were able to label a caspase 3 substrate CP18 with 18 F by click chemistry. Preliminary studies demonstrate that uptake of this tracer in aortic plaque strongly correlates with high levels of apoptotic activity by TUNEL staining and macrophage entry detected by CD68 staining. 18 F-CP18 may be a valuable imaging agent to detect plaque apoptosis and may allow early detection of hig-risk plaque.
Chemiluminescent nitrogen detection for HPLC is an important new high throughput technique for measuring yields and purities in synthetic organic chemistry. This destructive detector measures the ...total nitrogen content of a chromatographic peak, can be calibrated with any nitrogen containing compound, and thus allows quantitative analysis when authentic reference standards are not available. The application to solid phase synthesis is illustrated with a diketopiperazine synthesis.
HPLC/CLND is a technique for quantifying yields and purities in organic chemistry, which will be especially useful in solid phase synthesis and combinatorial chemistry
Purpose
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,
18
FJNJ-64413739 has been recently disclosed.
Procedures
We evaluated
18
FJNJ-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
18
FJNJ-64413739 was confirmed by performing blocking experiments with the P2X7 antagonist JNJ-54175446.
Results
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
18
FJNJ-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
18
FJNJ-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.
Conclusions
While further work is needed to study
18
FJNJ-64413739 in other types of neuroinflammation, the current results favorably characterize
18
FJNJ-64413739 as a potential PET tracer of central neuroinflammation.
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