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Frontiers in Pharmacology 2022Zapnometinib is a MEK inhibitor currently under clinical development for the treatment of COVID-19 and influenza. Zapnometinib has both antiviral and immunomodulatory...
Zapnometinib is a MEK inhibitor currently under clinical development for the treatment of COVID-19 and influenza. Zapnometinib has both antiviral and immunomodulatory effects. Information concerning the absorption, distribution, metabolism, and excretion of the compound following single oral doses of 30 mg/kg [C]-zapnometinib to rats was required to support pharmacology and toxicology studies in animals and clinical studies in man. As part of the development and safety assessment of this substance, zapnometinib was radioactively labeled and used for the investigation of time-dependent plasma concentrations, the rates and routes of excretion, the extent and time-course of compound distribution in body tissues, the metabolite profiles in plasma, urine and feces and the chemical nature of its metabolites. The present study reveals a rapid but low absorption of zapnometinib from the gastrointestinal tract, with more than 90% of the compound being excreted within 48 h, mainly feces. Whole body autoradiography confirms that zapnometinib was rapidly and widely distributed, with greatest concentrations in the circulatory and visceral tissues. Maximum plasma and tissue concentrations occurred between two and 8 h post dose. Penetration into the brain was low, and elimination from most tissues almost complete after 168 h. Metabolic profiles showed that the main clearance routes were metabolism oxidative reactions and glucuronidation. These results further strengthen the knowledge of zapnometinib with respect to the clinical development of the drug.
PubMed: 36545320
DOI: 10.3389/fphar.2022.1050193 -
EJNMMI Radiopharmacy and Chemistry Oct 2023Receptor interacting protein kinase 1 (RIPK1) is a serine/threonine kinase, which regulates programmed cell death and inflammation. Recently, the involvement of RIPK1 in...
BACKGROUND
Receptor interacting protein kinase 1 (RIPK1) is a serine/threonine kinase, which regulates programmed cell death and inflammation. Recently, the involvement of RIPK1 in the pathophysiology of Alzheimer's disease (AD) has been reported; RIPK1 is involved in microglia's phenotypic transition to their dysfunctional states, and it is highly expressed in the neurons and microglia in the postmortem brains in AD patients. They prompt neurodegeneration leading to accumulations of pathological proteins in AD. Therefore, regulation of RIPK1 could be a potential therapeutic target for the treatment of AD, and in vivo imaging of RIPK1 may become a useful modality in studies of drug discovery and pathophysiology of AD. The purpose of this study was to develop a suitable radioligand for positron emission tomography (PET) imaging of RIPK1.
RESULTS
(S)-2,2-dimethyl-1-(5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)propan-1-one (GSK'963) has a high affinity, selectivity for RIPK1, and favorable physiochemical properties based on its chemical structure. In this study, since C-labeling (half-life: 20.4 min) GSK'963 retaining its structure requiring the Grignard reaction of tert-butylmagnesium halides and [C]carbon dioxide was anticipated to give a low yield, we decided instead to C-label a GSK'963 analog ((S)-2,2-dimethyl-1-(5-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)propan-1-one, GG502), which has a high RIPK1 inhibitory activity equivalent to that of the original compound GSK'963. Thus, we successfully C-labeled GG502 using a Pd-mediated cross-coupling reaction in favorable yields (3.6 ± 1.9%) and radiochemical purities (> 96%), and molar activity (47-115 GBq/μmol). On autoradiography, radioactivity accumulation was observed for [C]GG502 and decreased by non-radioactive GG502 in the mouse spleen and human brain, indicating the possibility of specific binding of this ligand to RIPK1. On brain PET imaging in a rhesus monkey, [C]GG502 showed a good brain permeability (peak standardized uptake value (SUV) ~3.0), although there was no clear evidence of specific binding of [C]GG502. On brain PET imaging in acute inflammation model rats, [C]GG502 also showed a good brain permeability, and no significant increased uptake was observed in the lipopolysaccharide-treated side of striatum. On metabolite analysis in rats at 30 min after administration of [C]GG502, ~55% and ~10% of radioactivity was from unmetabolized [C]GG502 in the brain and the plasma, respectively.
CONCLUSIONS
We synthesized and evaluated a C-labeled PET ligand based on the methylated analog of GSK'963 for imaging of RIPK1 in the brain. Although in autoradiography of the resulting [C]GG502 indicated the possibility of specific binding, the actual PET imaging failed to detect any evidence of specific binding to RIPK1 despite its good brain permeability. Further development of radioligands with a higher binding affinity for RIPK1 in vivo and more stable metabolite profiles compared with the current compound may be required.
PubMed: 37853253
DOI: 10.1186/s41181-023-00217-z -
Journal of the American College of... Sep 2019Arterial fluorodeoxyglucose (FDG) positron emission tomography (PET) is considered a measure of atherosclerotic plaque macrophages and is used for quantification of...
BACKGROUND
Arterial fluorodeoxyglucose (FDG) positron emission tomography (PET) is considered a measure of atherosclerotic plaque macrophages and is used for quantification of disease activity in clinical trials, but the distribution profile of FDG across macrophages and other arterial cells has not been fully clarified.
OBJECTIVES
The purpose of this study was to analyze FDG uptake in different arterial tissues and their contribution to PET signal in normal and atherosclerotic arteries.
METHODS
Wild-type and D374Y-PCSK9 transgenic Yucatan minipigs were fed a high-fat, high-cholesterol diet to induce atherosclerosis and subjected to a clinical FDG-PET and computed tomography scan protocol. Volumes of arterial media, intima/lesion, macrophage-rich, and hypoxic tissues were measured in serial histological sections. Distributions of FDG in macrophages and other arterial tissues were quantified using modeling of the in vivo PET signal. In separate transgenic minipigs, the intra-arterial localization of FDG was determined directly by autoradiography.
RESULTS
Arterial FDG-PET signal appearance and intensity were similar to human imaging. The modeling approach showed high accuracy in describing the FDG-PET signal and revealed comparable FDG accumulation in macrophages and other arterial tissues, including medial smooth muscle cells. These findings were verified directly by autoradiography of normal and atherosclerotic arteries.
CONCLUSIONS
FDG is taken up comparably in macrophage-rich and -poor arterial tissues in minipigs. This offers a mechanistic explanation to a growing number of observations in clinical imaging studies that have been difficult to reconcile with macrophage-selective FDG uptake.
Topics: Animals; Arteries; Atherosclerosis; Female; Fluorodeoxyglucose F18; Male; Positron-Emission Tomography; Radiopharmaceuticals; Swine; Swine, Miniature; Tissue Distribution
PubMed: 31466620
DOI: 10.1016/j.jacc.2019.06.057 -
Cancer Immunology Research Sep 2022Immune checkpoint inhibitors (ICI) have been effective in treating a subset of refractory solid tumors, but only a small percentage of treated patients benefit from...
Immune checkpoint inhibitors (ICI) have been effective in treating a subset of refractory solid tumors, but only a small percentage of treated patients benefit from these therapies. Thus, there is a clinical need for reliable tools that allow for the early assessment of response to ICIs, as well as a preclinical need for imaging tools that aid in the future development and understanding of immunotherapies. Here we demonstrate that CD69, a canonical early-activation marker expressed on a variety of activated immune cells, including cytotoxic T cells and natural killer (NK) cells, is a promising biomarker for the early assessment of response to immunotherapies. We have developed a PET probe by radiolabeling a highly specific CD69 mAb, H1.2F3, with Zirconium-89 (89Zr), [89Zr]-deferoxamine (DFO)-H1.2F3. [89Zr]-DFO-H1.2F3 detected changes in CD69 expression on primary mouse T cells in vitro and detected activated immune cells in a syngeneic tumor immunotherapy model. In vitro uptake studies with [89Zr]-DFO-H1.2F3 showed a 15-fold increase in CD69 expression for activated primary mouse T cells, relative to untreated resting T cells. In vivo PET imaging showed that tumors of ICI-responsive mice had greater uptake than the tumors of nonresponsive and untreated mice. Ex vivo biodistribution, autoradiography, and IHC analyses supported the PET imaging findings. These data suggest that the CD69 PET imaging approach detects CD69 expression with sufficient sensitivity to quantify immune cell activation in a syngeneic mouse immunotherapy model and could allow for the prediction of therapeutic immune responses to novel immunotherapies.
Topics: Animals; Cell Line, Tumor; Deferoxamine; Immunologic Factors; Immunotherapy; Mice; Positron-Emission Tomography; Radioisotopes; Tissue Distribution; Zirconium
PubMed: 35862229
DOI: 10.1158/2326-6066.CIR-21-0874 -
Journal For Immunotherapy of Cancer Oct 2020To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal...
BACKGROUND
To better predict response to immune checkpoint therapy and toxicity in healthy tissues, insight in the in vivo behavior of immune checkpoint targeting monoclonal antibodies is essential. Therefore, we aimed to study in vivo pharmacokinetics and whole-body distribution of zirconium-89 (Zr) labeled programmed cell death protein-1 (PD-1) targeting pembrolizumab with positron-emission tomography (PET) in humanized mice.
METHODS
Humanized (huNOG) and non-humanized NOG mice were xenografted with human A375M melanoma cells. PET imaging was performed on day 7 post Zr-pembrolizumab (10 µg, 2.5 MBq) administration, followed by ex vivo biodistribution studies. Other huNOG mice bearing A375M tumors received a co-injection of excess (90 µg) unlabeled pembrolizumab or Zr-IgG control (10 µg, 2.5 MBq). Tumor and spleen tissue were studied with autoradiography and immunohistochemically including PD-1.
RESULTS
PET imaging and biodistribution studies showed high Zr-pembrolizumab uptake in tissues containing human immune cells, including spleen, lymph nodes and bone marrow. Tumor uptake of Zr-pembrolizumab was lower than uptake in lymphoid tissues, but higher than uptake in other organs. High uptake in lymphoid tissues could be reduced by excess unlabeled pembrolizumab. Tracer activity in blood pool was increased by addition of unlabeled pembrolizumab, but tumor uptake was not affected. Autoradiography supported PET findings and immunohistochemical staining on spleen and lymph node tissue showed PD-1 positive cells, whereas tumor tissue was PD-1 negative.
CONCLUSION
Zr-pembrolizumab whole-body biodistribution showed high PD-1-mediated uptake in lymphoid tissues, such as spleen, lymph nodes and bone marrow, and modest tumor uptake. Our data may enable evaluation of Zr-pembrolizumab whole-body distribution in patients.
Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Immunological; Biomarkers, Tumor; Cell Line, Tumor; Humans; Immunotherapy; Mice; Programmed Cell Death 1 Receptor; Tertiary Lymphoid Structures
PubMed: 33020241
DOI: 10.1136/jitc-2020-000938 -
Frontiers in Immunology 2022Increased glutamine metabolism by macrophages is associated with development of atherosclerotic lesions. Positron emission tomography/computed tomography (PET/CT) with a...
Increased glutamine metabolism by macrophages is associated with development of atherosclerotic lesions. Positron emission tomography/computed tomography (PET/CT) with a glutamine analog (2S,4)-4-F-fluoroglutamine (F-FGln) allows quantification of glutamine consumption . Here, we investigated uptake of F-FGln by atherosclerotic lesions in mice and compared the results with those obtained using the glucose analog 2-deoxy-2-F-fluoro--glucose (F-FDG). Uptake of F-FGln and F-FDG by healthy control mice (C57BL/6JRj) and atherosclerotic low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLRApoB) was investigated. The mice were injected intravenously with F-FGln or F-FDG for PET/CT imaging. After sacrifice at 70 minutes post-injection, tracer uptake was analyzed by gamma counting of excised tissues and by autoradiography of aorta cryosections, together with histological and immunohistochemical analyses. We found that myocardial uptake of F-FGln was low. PET/CT detected lesions in the aortic arch, with a target-to-background ratio (SUV, aortic arch/SUV, blood) of 1.95 ± 0.42 (mean ± standard deviation). Gamma counting revealed that aortic uptake of F-FGln by LDLRApoB mice (standardized uptake value [SUV], 0.35 ± 0.06) was significantly higher than that by healthy controls (0.20 ± 0.08, = 0.03). More detailed analysis by autoradiography revealed that the plaque-to-healthy vessel wall ratio of F-FGln (2.90 ± 0.42) was significantly higher than that of F-FDG (1.93 ± 0.22, = 0.004). Immunohistochemical staining confirmed that F-FGln uptake in plaques co-localized with glutamine transporter SLC7A7-positive macrophages. Collectively these data show that the F-FGln PET tracer detects inflamed atherosclerotic lesions. Thus, exploiting glutamine consumption using F-FGln PET may have translational relevance for studying atherosclerotic inflammation.
Topics: Animals; Apolipoprotein B-100; Atherosclerosis; Disease Models, Animal; Fluorodeoxyglucose F18; Glutamine; Male; Mice; Mice, Inbred C57BL; Plaque, Atherosclerotic; Positron Emission Tomography Computed Tomography; Receptors, LDL
PubMed: 35145523
DOI: 10.3389/fimmu.2022.821423 -
Translational Psychiatry Jan 2021The serotonin 5-HT receptor has attracted wide attention as a target for treatment of psychiatric disorders. Although this receptor is important in the pharmacological... (Review)
Review
The serotonin 5-HT receptor has attracted wide attention as a target for treatment of psychiatric disorders. Although this receptor is important in the pharmacological mechanisms of action of new-generation antipsychotics, its characterization remains incomplete. Studies based on in vitro molecular imaging on brain tissue by autoradiography, and more recently in vivo PET imaging, have not yielded clear results, in particular due to the limitations of current 5-HT radiotracers, which lack specificity and/or bind to all 5-HT receptors, regardless of their functional status. The new concept of PET neuroimaging of functionally active G-protein-coupled receptors makes it possible to revisit PET brain exploration by enabling new research paradigms. For the 5-HT receptor it is now possible to use [F]-F13640, a 5-HT receptor radioligand with high efficacy agonist properties, to specifically visualize and quantify functionally active receptors, and to relate this information to subjects' pathophysiological or pharmacological state. We therefore propose imaging protocols to follow changes in the pattern of functional 5-HT receptors in relation to mood deficits or cognitive processes. This could allow improved discrimination of different schizophrenia phenotypes and greater understanding of the basis of therapeutic responses to antipsychotic drugs. Finally, as well as targeting functionally active receptors to gain insights into the role of 5-HT receptors, the concept can also be extended to the study of other receptors involved in the pathophysiology or therapy of psychiatric disorders.
Topics: Autoradiography; Brain; Humans; Receptor, Serotonin, 5-HT1A; Schizophrenia; Serotonin; Serotonin 5-HT1 Receptor Agonists
PubMed: 33414418
DOI: 10.1038/s41398-020-01119-3 -
Journal of Developmental Biology Dec 2022The adhesive digital pads in some gecko and anoline lizards are continuously utilized for movements on vertical surfaces that may determine wear and a decrease of... (Review)
Review
The adhesive digital pads in some gecko and anoline lizards are continuously utilized for movements on vertical surfaces that may determine wear and a decrease of adhesion efficiency. The pads are formed by lamellae bearing adhesive setae that are worn out following frequent usage and are replaced by new inner setae that maintain an efficient adhesion. Whether the extensive usage of adhesive setae determines a higher shedding frequency in the digital pads with respect to other body regions remains unknown. Setae replacement has been analyzed in embryos and adult lizards using autoradiography and 5BrdU-immunohistochemistry. The observation strongly suggests that during development and epidermal renewal in adult lamellae, there is a shifting of the outer setae toward the apex of the lamella. This movement is likely derived from the continuous addition of proteins in the beta- and alpha-layers sustaining the outer setae while the inner setae are forming. Ultrastructural and in situ hybridization studies indicate that the thin outer beta- and alpha-layers still contain mRNAs and ribosomes that may contribute to the continuous production of corneous beta proteins (CBPs) and keratins for the growth of the free margin at the apex of the lamella. This process determines the apical shifting and release of the old setae, while the new inner setae formed underneath becomes the new outer setae.
PubMed: 36648905
DOI: 10.3390/jdb11010003 -
Communications Biology Oct 2022Protein degradation mediated by the ubiquitin-proteasome pathway regulates signaling events in many physiological and pathological conditions. In vitro degradation...
Protein degradation mediated by the ubiquitin-proteasome pathway regulates signaling events in many physiological and pathological conditions. In vitro degradation assays have been instrumental in the understanding of how cell proliferation and other fundamental cellular processes are regulated. These assays are direct, time-specific and highly informative but also laborious, typically relying on low-throughput polyacrylamide gel-electrophoresis followed by autoradiography or immunoblotting. We present protein degradation on chip (pDOC), a MITOMI-based integrated microfluidic technology for discovery and analysis of proteins degradation in cell-free extracts. The platform accommodates hundreds of microchambers on which protein degradation is assayed quickly, simultaneously and using minute amounts of reagents in one or many physiochemical environments. Essentially, pDOC provides a sensitive multiplex alternative to the conventional degradation assay, with relevance to biomedical and translational research associated with regulated proteolysis.
Topics: Microfluidics; Proteolysis; Cell Extracts; Electrophoresis, Polyacrylamide Gel; Immunoblotting
PubMed: 36307570
DOI: 10.1038/s42003-022-04103-3 -
Arteriosclerosis, Thrombosis, and... Aug 2022Patients with thoracic aortopathy are at increased risk of catastrophic aortic dissection, carrying with it substantial mortality and morbidity. Although granular medial...
BACKGROUND
Patients with thoracic aortopathy are at increased risk of catastrophic aortic dissection, carrying with it substantial mortality and morbidity. Although granular medial calcinosis (medial microcalcification) has been associated with thoracic aortopathy, its relationship to disease severity has yet to be established.
METHODS
One hundred one thoracic aortic specimens were collected from 57 patients with thoracic aortopathy and 18 control subjects. Standardized histopathologic scores, immunohistochemistry, and nanoindentation (tissue elastic modulus) were compared with the extent of microcalcification on von Kossa histology and 18F-sodium fluoride autoradiography.
RESULTS
Microcalcification content was higher in thoracic aortopathy samples with mild (n=28; 6.17 [2.71-10.39]; ≤0.00010) or moderate histopathologic degeneration (n=30; 3.74 [0.87-11.80]; <0.042) compared with control samples (n=18; 0.79 [0.36-1.90]). Alkaline phosphatase (n=26; =0.0019) and OPN (osteopontin; n=26; =0.0045) staining were increased in tissue with early aortopathy. Increasingly severe histopathologic degeneration was related to reduced microcalcification (n=82; Spearman ρ, -0.51; <0.0001)-a process closely linked with elastin loss (n=82; Spearman ρ, -0.43; <0.0001) and lower tissue elastic modulus (n=28; Spearman ρ, 0.43; =0.026).F-sodium fluoride autoradiography demonstrated good correlation with histologically quantified microcalcification (n=66; r=0.76; <0.001) and identified areas of focal weakness in vivo.
CONCLUSIONS
Medial microcalcification is a marker of aortopathy, although progression to severe aortopathy is associated with loss of both elastin fibers and microcalcification.F-sodium fluoride positron emission tomography quantifies medial microcalcification and is a feasible noninvasive imaging modality for identifying aortic wall disruption with major translational promise.
Topics: Aorta; Calcinosis; Elastin; Humans; Severity of Illness Index; Sodium Fluoride
PubMed: 35770666
DOI: 10.1161/ATVBAHA.122.317111