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Nuclear Medicine and Biology 2022Receptor-interacting protein kinase 1 (RIPK1) has emerged as a crucial regulator of necroptosis and the inflammatory response by activating a group of downstream immune...
INTRODUCTION
Receptor-interacting protein kinase 1 (RIPK1) has emerged as a crucial regulator of necroptosis and the inflammatory response by activating a group of downstream immune receptors. It has been recognized as a pivotal contributor to cell death and inflammation in various physiological and pathological processes. RIPK1 deficiency or dysregulation in humans can cause severe immunodeficiency and neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis. Recently, diverse structures of RIPK1 inhibitors have been developed as potential therapeutics for neurodegenerative diseases and other pathological inflammatory processes. 7-oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine (Compound 5 or TZ7774) was reported as a novel RIPK1 inhibitor with a K of 0.91 nM that can suppress necroptosis in mouse and human cells. To develop a radiotracer for investigating the RIPK1 in vivo, we radiosynthesized [C]TZ7774 and performed preliminary in vitro and in vivo evaluations in rodents and macaque.
METHODS
Synthesis of the desmethyl precursor TZ7790 was performed and optimized. The radiosynthesis of [C]TZ7774 was achieved through TZ7790 reacting with [C]methyl iodide via N-methylation. Ex vivo biodistribution of [C]TZ7774 was performed in normal Sprague-Dawley rats. Characterization of [C]TZ7774 in response to inflammation was performed using ex vivo biodistribution study in normal and LPS treated (10 mg/kg) C57BL/6 mice, and in vitro autoradiography and immunohistochemistry of the spleen. MicroPET brain study of [C]TZ7774 in the macaque was also performed.
RESULTS AND CONCLUSIONS
The radiosynthesis of [C]TZ7774 was achieved with good radiochemical yield (30-40%, decay corrected to the end of bombardment (EOB)), high chemical purity (>90%), high radiochemical purity (>99%), and high molar activity (>207 GBq/μmol, decay corrected to EOB). Biodistribution studies in Sprague-Dawley rats showed [C]TZ7774 has a high brain uptake of 0.53 (%ID/g) at 5 min post injection; pancreas, spleen, kidney, and liver also showed a relatively high initial uptake of 0.49, 0.41, 0.62, and 0.95 at 5 min respectively. Uptake of [C]TZ7774 increased in LPS-treated C57BL/6 mice by 40.9%, 90.4%, and 54.9% in liver, spleen, and kidney respectively. In vitro autoradiography study also revealed increased uptake of [C]TZ7774 in the spleen of LPS-treated mice. Further characterization with immunohistochemistry confirmed increased expression of RIPK1 in red and white pulp of the spleen for mice pre-treated with LPS. MicroPET demonstrated that [C]TZ7774 had good initial brain uptake in macaque with an (SUV) of ∼3.7 at 6-10 min, and quickly washed out from brain. These data confirm successful radiosynthesis of a RIPK1 specific radiotracer [C]TZ7774. Our preliminary studies showed good response to LPS-induced inflammation in rodents and good uptake in macaque brain. [C]TZ7774 has a potential to image RIPK1 related necroptosis and inflammatory processes.
Topics: Animals; Brain; Carbon Radioisotopes; Inflammation; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Neurodegenerative Diseases; Positron-Emission Tomography; Protein Kinases; Rats; Rats, Sprague-Dawley; Tissue Distribution
PubMed: 35472678
DOI: 10.1016/j.nucmedbio.2022.04.006 -
Journal of Nuclear Medicine : Official... Mar 2023Chronic traumatic encephalopathy (CTE) is a neurologic disorder associated with head injuries, diagnosed by the perivascular accumulation of hyperphosphorylated tau...
Chronic traumatic encephalopathy (CTE) is a neurologic disorder associated with head injuries, diagnosed by the perivascular accumulation of hyperphosphorylated tau protein (phospho-tau) identified at autopsy. Tau PET radiopharmaceuticals developed for imaging Alzheimer disease are under evaluation for brain injuries. The goal of this study was to conduct a head-to-head in vitro evaluation of 5 tau PET radiotracers in subjects pathologically diagnosed with CTE. Autoradiography was used to assess the specific binding and distribution of H-flortaucipir (also known as Tauvid, AV-1451, and T807), H-MK-6240 (also known as florquinitau), H-PI-2620, H-APN-1607 (also known as PM-PBB3 and florzolotau), and H-CBD-2115 (also known as H-OXD-2115) in fresh-frozen human postmortem CTE brain tissue (stages I-IV). Immunohistochemistry was performed for phospho-tau with AT8, 3R tau with RD3, 4R tau with RD4 and amyloid-β with 6F/3D antibodies. Tau target density (maximum specific binding) was quantified by saturation analysis with H-flortaucipir in tissue sections. H-flortaucipir demonstrated a positive signal in all CTE cases examined, with varying degrees of specific binding (68.7% ± 10.5%; = 12) defined by homologous blockade and to a lesser extent by heterologous blockade with MK-6240 (27.3% ± 13.6%; = 12). The H-flortaucipir signal was also displaced by the monoamine oxidase (MAO)-A inhibitor clorgyline (43.9% ± 4.6%; = 3), indicating off-target binding to MAO-A. H-APN-1607 was moderately displaced in homologous blocking studies and was not displaced by H-flortaucipir; however, substantial displacement was observed when blocking with the β-amyloid-targeting compound NAV-4694. H-MK-6240 and H-PI-2620 had negligible binding in all but 2 CTE IV cases, and binding may be attributed to pathology severity or mixed Alzheimer disease/CTE pathology. H-CBD-2115 showed moderate binding, displaced under homologous blockade, and aligned with 4R-tau immunostaining. In human CTE tissues, H-flortaucipir and H-APN-1607 revealed off-target binding to MAO-A and amyloid-β, respectively, and should be considered if these radiotracers are used in PET imaging studies of patients with brain injuries. H-MK-6240 and H-PI-2620 bind to CTE tau in severe- or mixed-pathology cases, and their respective F PET radiotracers warrant further evaluation in patients with severe suspected CTE.
Topics: Humans; tau Proteins; Chronic Traumatic Encephalopathy; Alzheimer Disease; Amyloid beta-Peptides; Brain Injuries
PubMed: 36109185
DOI: 10.2967/jnumed.122.264404 -
Scientific Reports Dec 2021Biomarkers for the measurement of islets of Langerhans could help elucidate the etiology of diabetes. Synaptic vesicle glycoprotein 2 A (SV2A) is a potential marker...
Biomarkers for the measurement of islets of Langerhans could help elucidate the etiology of diabetes. Synaptic vesicle glycoprotein 2 A (SV2A) is a potential marker reported to be localized in the endocrine pancreas. [C]UCB-J is a novel positron emission tomography (PET) radiotracer that binds to SV2A and was previously evaluated as a synaptic marker in the central nervous system. Here, we evaluated whether [C]UCB-J could be utilized as a PET tracer for the islets of Langerhans in the pancreas by targeting SV2A. The mRNA transcription of SV2A was evaluated in human isolated islets of Langerhans and exocrine tissue. In vitro autoradiography was performed on pancreas and brain sections from rats and pigs, and consecutive sections were immunostained for insulin. Sprague-Dawley rats were examined with PET-MRI and ex vivo autoradiography at baseline and with administration of levetiracetam (LEV). Similarly, pigs were examined with dynamic PET-CT over the pancreas and brain after administration of [C]UCB-J at baseline and after pretreatment with LEV. In vivo radioligand binding was assessed using a one-compartment tissue model. The mRNA expression of SV2A was nearly 7 times higher in endocrine tissue than in exocrine tissue (p < 0.01). In vitro autoradiography displayed focal binding of [C]UCB-J in the pancreas of rats and pigs, but the binding pattern did not overlap with the insulin-positive areas or with ex vivo autoradiography. In rats, pancreas binding was higher than that in negative control tissues but could not be blocked by LEV. In pigs, the pancreas and brain exhibited accumulation of [C]UCB-J above the negative control tissue spleen. While brain binding could be blocked by pretreatment with LEV, a similar effect was not observed in the pancreas. Transcription data indicate SV2A to be a valid target for imaging islets of Langerhans, but [C]UCB-J does not appear to have sufficient sensitivity for this application.
Topics: Animals; Female; Islets of Langerhans; Male; Membrane Glycoproteins; Nerve Tissue Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Pyridines; Pyrrolidinones; Radiopharmaceuticals; Rats, Sprague-Dawley; Swine; Rats
PubMed: 34963683
DOI: 10.1038/s41598-021-04188-6 -
European Journal of Nuclear Medicine... Jun 2021Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to...
PURPOSE
Deposition of misfolded alpha-synuclein (αSYN) aggregates in the human brain is one of the major hallmarks of synucleinopathies. However, a target-specific tracer to detect pathological aggregates of αSYN remains lacking. Here, we report the development of a positron emission tomography (PET) tracer based on anle138b, a compound shown to have therapeutic activity in animal models of neurodegenerative diseases.
METHODS
Specificity and selectivity of [H]MODAG-001 were tested in in vitro binding assays using recombinant fibrils. After carbon-11 radiolabeling, the pharmacokinetic and metabolic profile was determined in mice. Specific binding was quantified in rats, inoculated with αSYN fibrils and using in vitro autoradiography in human brain sections of Lewy body dementia (LBD) cases provided by the Neurobiobank Munich (NBM).
RESULTS
[H]MODAG-001 revealed a very high affinity towards pure αSYN fibrils (K = 0.6 ± 0.1 nM) and only a moderate affinity to hTau46 fibrils (K = 19 ± 6.4 nM) as well as amyloid-β fibrils (K = 20 ± 10 nM). [C]MODAG-001 showed an excellent ability to penetrate the mouse brain. Metabolic degradation was present, but the stability of the parent compound improved after selective deuteration of the precursor. (d)-[C]MODAG-001 binding was confirmed in fibril-inoculated rat striata using in vivo PET imaging. In vitro autoradiography showed no detectable binding to aggregated αSYN in human brain sections of LBD cases, most likely, because of the low abundance of aggregated αSYN against background protein.
CONCLUSION
MODAG-001 provides a promising lead structure for future compound development as it combines a high affinity and good selectivity in fibril-binding assays with suitable pharmacokinetics and biodistribution properties.
Topics: Animals; Carbon Radioisotopes; Lewy Body Disease; Mice; Neurodegenerative Diseases; Positron-Emission Tomography; Radiopharmaceuticals; Rats; Tissue Distribution; alpha-Synuclein
PubMed: 33369690
DOI: 10.1007/s00259-020-05133-x -
Scientific Reports Sep 2021Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The...
Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer's disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer's disease patients.
Topics: Alzheimer Disease; Animals; Autoradiography; Brain; Disease Models, Animal; Humans; Huntingtin Protein; Huntington Disease; Immunohistochemistry; Mice; Mice, Transgenic; Nitrogen Radioisotopes; Positron-Emission Tomography; Protein Aggregates; Protein Aggregation, Pathological; Radioactive Tracers; Radioligand Assay; Radiopharmaceuticals; Recombinant Proteins
PubMed: 34504195
DOI: 10.1038/s41598-021-97334-z -
ACS Chemical Neuroscience Feb 2022The homo-pentameric alpha 7 receptor is one of the major types of neuronal nicotinic acetylcholine receptors (α7-nAChRs) related to cognition, memory formation, and...
The homo-pentameric alpha 7 receptor is one of the major types of neuronal nicotinic acetylcholine receptors (α7-nAChRs) related to cognition, memory formation, and attention processing. The mapping of α7-nAChRs by PET pulls a lot of attention to realize the mechanism and development of CNS diseases such as AD, PD, and schizophrenia. Several PET radioligands have been explored for the detection of the α7-nAChR. F-ASEM is the most functional for quantification of α7-nAChRs in the human brain. The first aim of this study was to initially use results from in silico and machine learning techniques to prescreen and predict the binding energy and other properties of ASEM analogues and to interpret these properties in terms of atomic structures using F-ASEM as a lead structure, and second, to label some selected candidates with carbon-11/hydrogen-3 (C/H) and to evaluate the binding properties and using the labeled candidates. In silico predictions are obtained from perturbation free-energy calculations preceded by molecular docking, molecular dynamics, and metadynamics simulations. Machine learning techniques have been applied for the BBB and P-gp-binding properties. Six analogues of ASEM were labeled with C, and three of them were additionally labeled with H. Binding properties were further evaluated using autoradiography (ARG) and PET measurements in non-human primates (NHPs). Radiometabolites were measured in NHP plasma. All six compounds were successfully synthesized. Evaluation with ARG showed that C-Kln83 was preferably binding to the α7-nAChR. Competition studies showed that 80% of the total binding was displaced. Further ARG studies using H-KIn-83 replicated the preliminary results. In the NHP PET study, the distribution pattern of C-KIn-83 was similar to other α7 nAChR PET tracers. The brain uptake was relatively low and increased by the administration of tariquidar, indicating a substrate of P-gp. The ASEM blocking study showed that C-KIn-83 specifically binds to α7 nAChRs. Preliminary evaluation of KIn-83 by ARG with both C and H and evaluation in NHP showed favorable properties for selectively imaging α7-nAChRs, despite a relatively low brain uptake.
Topics: Animals; Azabicyclo Compounds; Cyclic S-Oxides; Molecular Docking Simulation; Positron-Emission Tomography; Receptors, Nicotinic; alpha7 Nicotinic Acetylcholine Receptor
PubMed: 35020351
DOI: 10.1021/acschemneuro.1c00730 -
Arteriosclerosis, Thrombosis, and... Sep 2023F-GP1 is a novel positron-emitting radiotracer that is highly specific for activated platelets and thrombus. In a proof-of-concept study, we aimed to determine its...
BACKGROUND
F-GP1 is a novel positron-emitting radiotracer that is highly specific for activated platelets and thrombus. In a proof-of-concept study, we aimed to determine its potential clinical application in establishing the role and origin of thrombus in ischemic stroke.
METHODS
Eleven patients with recent ischemic stroke (n=9) or transient ischemic attack (n=2) underwent F-GP1 positron emission tomography and computed tomography angiography at a median of 11 (range, 2-21) days from symptom onset. F-GP1 uptake (maximum target-to-background ratio) was assessed in the carotid arteries and brain.
RESULTS
F-GP1 uptake was identified in 10 of 11 patients: 4 in the carotid arteries only, 3 in the brain only, and 3 in both the brain and carotid arteries. In those with carotid uptake, 4 participants had >50% stenosis and 3 had nonstenotic disease. One case had bilateral stenotic disease (>70%), but only the culprit carotid artery demonstrated F-GP1 uptake. The average uptake was higher in the culprit (median maximum target-to-background ratio, 1.55 [interquartile range, 1.26-1.82]) compared with the contralateral nonculprit carotid artery (maximum target-to-background ratio, 1.22 [1.19-1.6]). In those with brain F-GP1 uptake (maximum target-to-background ratio, 6.45 [4.89-7.65]), areas of acute infarction on computed tomography correlated with brain F-GP1 uptake in 6 cases. Ex vivo autoradiography of postmortem infarcted brain tissue showed focal uptake corresponding to intraluminal thrombus within the culprit vessel and downstream microvasculature. There was also evidence of diffuse uptake within some of the infarcted brain tissue reflecting parenchymal petechial hemorrhage.
CONCLUSIONS
F-GP1 positron emission tomography and computed tomography angiography is a novel noninvasive method of identifying in vivo cerebrovascular thrombosis, which holds major promise in understanding the role and origin of thrombosis in stroke.
REGISTRATION
URL: https://www.
CLINICALTRIALS
gov; Unique identifier: NCT03943966.
Topics: Humans; Carotid Arteries; Carotid Stenosis; Ischemic Attack, Transient; Ischemic Stroke; Stroke; Thrombosis
PubMed: 37439259
DOI: 10.1161/ATVBAHA.122.318204 -
Journal of Nuclear Medicine : Official... Apr 2022Digital autoradiography (DAR) is a powerful tool to quantitatively determine the distribution of a radiopharmaceutical within a tissue section and is widely used in drug...
Digital autoradiography (DAR) is a powerful tool to quantitatively determine the distribution of a radiopharmaceutical within a tissue section and is widely used in drug discovery and development. However, the low image resolution and significant background noise can result in poor correlation, even errors, between radiotracer distribution, anatomic structure, and molecular expression profiles. Differing from conventional optical systems, the point-spread function in DAR is determined by properties of radioisotope decay, phosphor, and digitizer. Calibration of an experimental point-spread function a priori is difficult, prone to error, and impractical. We have developed a content-adaptive restoration algorithm to address these problems. We model the DAR imaging process using a mixed Poisson-gaussian model and blindly restore the image by a penalized maximum-likelihood expectation-maximization algorithm (PG-PEM). PG-PEM implements a patch-based estimation algorithm with density-based spatial clustering of applications with noise to estimate noise parameters and uses L2 and Hessian Frebonius norms as regularization functions to improve performance. First, PG-PEM outperformed other restoration algorithms at the denoising task ( 0.01). Next, we implemented PG-PEM on preclinical DAR images (F-FDG, treated mouse tumor and heart; F-NaF, treated mouse femur) and clinical DAR images (bone biopsy sections from RaCl-treated castration-resistant prostate cancer patients). DAR images restored by PG-PEM of all samples achieved a significantly higher effective resolution and contrast-to-noise ratio and a lower SD of background ( 0.0001). Additionally, by comparing the registration results between the clinical DAR images and the segmented bone masks from the corresponding histologic images, we found that the radiopharmaceutical distribution was significantly improved ( 0.0001). PG-PEM is able to increase resolution and contrast while robustly accounting for DAR noise and demonstrates the capacity to be widely implemented to improve preclinical and clinical DAR imaging of radiopharmaceutical distribution.
Topics: Algorithms; Animals; Autoradiography; Diagnostic Imaging; Fluorodeoxyglucose F18; Humans; Image Processing, Computer-Assisted; Male; Mice; Phantoms, Imaging; Radiopharmaceuticals; Tissue Distribution
PubMed: 34385337
DOI: 10.2967/jnumed.121.262270 -
Biomedicines Mar 2023High-resolution scans of immunohistochemical (IHC) stains of Alzheimer's disease (AD) brain slices and radioligand autoradiography both provide information about the...
Measurement of Aβ Amyloid Plaques and Tau Protein in Postmortem Human Alzheimer's Disease Brain by Autoradiography Using [F]Flotaza, [I]IBETA, [I]IPPI and Immunohistochemistry Analysis Using QuPath.
High-resolution scans of immunohistochemical (IHC) stains of Alzheimer's disease (AD) brain slices and radioligand autoradiography both provide information about the distribution of Aβ plaques and Tau, the two common proteinopathies in AD. Accurate assessment of the amount and regional location of Aβ plaques and Tau is essential to understand the progression of AD pathology. Our goal was to develop a quantitative method for the analysis of IHC-autoradiography images. Postmortem anterior cingulate (AC) and corpus callosum (CC) from AD and control (CN) subjects were IHC stained with anti-Aβ for Aβ plaques and autoradiography with [F]flotaza and [I]IBETA for Aβ plaques. For Tau, [I]IPPI, a new radiotracer, was synthesized and evaluated in the AD brain. For Tau imaging, brain slices were IHC stained with anti-Tau and autoradiography using [I]IPPI and [I]IPPI. Annotations for Aβ plaques and Tau using QuPath for training and pixel classifiers were generated to measure the percent of the area of Aβ plaques and Tau in each slice. The binding of [I]IPPI was observed in all AD brains with an AC/CC ratio > 10. Selectivity to Tau was shown by blocking [I]IPPI with MK-6240. Percent positivity for Aβ plaques was 4-15%, and for Tau, it was 1.3 to 35%. All IHC Aβ plaque-positive subjects showed [F]flotaza and [I]IBETA binding with a positive linear correlation (r > 0.45). Tau-positive subjects showed [I]IPPI binding with a stronger positive linear correlation (r > 0.80). This quantitative IHC-autoradiography approach provides an accurate measurement of Aβ plaques and Tau within and across subjects.
PubMed: 37189652
DOI: 10.3390/biomedicines11041033 -
Angiogenesis Aug 2023APJ has been extensively described in the pathophysiology of angiogenesis and cell proliferation. The prognostic value of APJ overexpression in many diseases is now...
APJ has been extensively described in the pathophysiology of angiogenesis and cell proliferation. The prognostic value of APJ overexpression in many diseases is now established. This study aimed to design a PET radiotracer that specifically binds to APJ. Apelin-F13A-NODAGA (AP747) was synthesized and radiolabeled with gallium-68 ([Ga]Ga-AP747). Radiolabeling purity was excellent (> 95%) and stable up to 2 h. Affinity constant of [Ga]Ga-AP747 was measured on APJ-overexpressing colon adenocarcinoma cells and was in nanomolar range. Specificity of [Ga]Ga-AP747 for APJ was evaluated in vitro by autoradiography and in vivo by small animal PET/CT in both colon adenocarcinoma mouse model and Matrigel plug mouse model. Dynamic of [Ga]Ga-AP747 PET/CT biodistributions was realized on healthy mice and pigs for two hours, and quantification of signal in organs showed a suitable pharmacokinetic profile for PET imaging, largely excreted by urinary route. Matrigel mice and hindlimb ischemic mice were submitted to a 21-day longitudinal follow-up with [Ga]Ga-AP747 and [Ga]Ga-RGD small animal PET/CT. [Ga]Ga-AP747 PET signal in Matrigel was significantly more intense than that of [Ga]Ga-RGD. Revascularization of the ischemic hind limb was followed by LASER Doppler. In the hindlimb, [Ga]Ga-AP747 PET signal was more than twice higher than that of [Ga]Ga-RGD on day 7, and significantly superior over the 21-day follow-up. A significant, positive correlation was found between the [Ga]Ga-AP747 PET signal on day 7 and late hindlimb perfusion on day 21. We developed a new PET radiotracer that specifically binds to APJ, [Ga]Ga-AP747 that showed more efficient imaging properties than the most clinically advanced tracer of angiogenesis, [Ga]Ga-RGD.
Topics: Animals; Mice; Swine; Apelin; Apelin Receptors; Gallium Radioisotopes; Positron Emission Tomography Computed Tomography; Adenocarcinoma; Colonic Neoplasms; Positron-Emission Tomography; Molecular Imaging; Oligopeptides
PubMed: 36973482
DOI: 10.1007/s10456-023-09875-8