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Advanced Drug Delivery Reviews May 2022The success of the messenger RNA-based COVID-19 vaccines of Moderna and Pfizer/BioNTech marks the beginning of a new chapter in modern medicine. However, the rapid rise... (Review)
Review
The success of the messenger RNA-based COVID-19 vaccines of Moderna and Pfizer/BioNTech marks the beginning of a new chapter in modern medicine. However, the rapid rise of mRNA therapeutics has resulted in a regulatory framework that is somewhat lagging. The current guidelines either do not apply, do not mention RNA therapeutics, or do not have widely accepted definitions. This review describes the guidelines for preclinical biodistribution studies of mRNA/siRNA therapeutics and highlights the relevant differences for mRNA vaccines. We also discuss the role of in vivo RNA imaging techniques and other assays to fulfill and/or complement the regulatory requirements. Specifically, quantitative whole-body autoradiography, microautoradiography, mass spectrometry-based assays, hybridization techniques (FISH, bDNA), PCR-based methods, in vivo fluorescence imaging, and in vivo bioluminescence imaging, are discussed. We conclude that this new and rapidly evolving class of medicines demands a multi-layered approach to fully understand its biodistribution and in vivo characteristics.
Topics: COVID-19; COVID-19 Vaccines; Humans; RNA, Messenger; RNA, Small Interfering; Tissue Distribution; mRNA Vaccines
PubMed: 35351470
DOI: 10.1016/j.addr.2022.114236 -
European Journal of Nuclear Medicine... Sep 2022Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor...
PURPOSE
Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies.
METHODS
FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule-based FAP-targeting agent.
RESULTS
Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of Ga-FAP-2286, In-FAP-2286, and Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46.
CONCLUSION
In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of Ga-FAP-2286 for imaging and Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.
Topics: Adult; Animals; Cell Line, Tumor; Fibroblasts; Gallium Radioisotopes; HEK293 Cells; Humans; Mice; Radionuclide Imaging; Sarcoma; Tissue Distribution; Tumor Microenvironment
PubMed: 35608703
DOI: 10.1007/s00259-022-05842-5 -
Journal of Nuclear Medicine : Official... Dec 2019Heart failure remains a major source of late morbidity and mortality after myocardial infarction (MI). The temporospatial presence of activated fibroblasts in the...
Heart failure remains a major source of late morbidity and mortality after myocardial infarction (MI). The temporospatial presence of activated fibroblasts in the injured myocardium predicts the quality of cardiac remodeling after MI. Therefore, monitoring of activated fibroblasts is of great interest for studying cardiac remodeling after MI. Fibroblast activation protein (FAP) expression is upregulated in activated fibroblasts. This study investigated the feasibility of imaging activated fibroblasts with a new Ga-labeled FAP inhibitor (Ga-FAPI-04) for PET imaging of fibroblast activation in a preclinical model of MI. MI and sham-operated rats were scanned with Ga-FAPI-04 PET/CT (1, 3, 6, 14, 23, and 30 d after MI) and with F-FDG (3 d after MI). Dynamic Ga-FAPI-04 PET and blocking studies were performed on MI rats 7 d after coronary ligation. After in vivo scans, the animals were euthanized and their hearts harvested for ex vivo analyses. Cryosections were prepared for autoradiography, hematoxylin and eosin (H&E), and immunofluorescence staining. Ga-FAPI-04 uptake in the injured myocardium peaked on day 6 after coronary ligation. The tracer accumulated intensely in the MI territory, as identified by decreased F-FDG uptake and confirmed by PET/MR and H&E staining. Autoradiography and H&E staining of cross-sections revealed that Ga-FAPI-04 accumulated mainly at the border zone of the infarcted myocardium. In contrast, there was only minimal uptake in the infarct of the blocked rats, comparable to the uptake in the remote noninfarcted myocardium (PET image-derived ratio of infarct uptake to remote uptake: 6 ± 2). Immunofluorescence staining confirmed the presence of FAP-positive myofibroblasts in the injured myocardium. Morphometric analysis of the whole-heart sections demonstrated 3- and 8-fold higher FAP-positive fibroblast density in the border zone than in the infarct center and remote area, respectively. Ga-FAPI-04 represents a promising radiotracer for in vivo imaging of post-MI fibroblast activation. Noninvasive imaging of activated fibroblasts may have significant diagnostic and prognostic value, which could aid clinical management of patients after MI.
Topics: Animals; Feasibility Studies; Fibroblasts; Gallium Radioisotopes; Isotope Labeling; Male; Membrane Proteins; Myocardial Infarction; Positron Emission Tomography Computed Tomography; Quinolines; Rats; Rats, Wistar
PubMed: 31405922
DOI: 10.2967/jnumed.119.226993 -
The Journal of Headache and Pain May 2023Cortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative...
BACKGROUND AND AIMS
Cortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative electrophysiological event underlying migraine aura and a headache trigger. Migraine is three times more prevalent in women than men, linked to circulating female hormones. High estrogen levels or estrogen withdrawal may be a migraine trigger for many women. We, therefore, aimed to examine whether sex, gonadectomy, and female hormone supplementation and withdrawal affect the susceptibility to CSD.
METHODS
To determine CSD susceptibility, we recorded the frequency of CSDs triggered during 2-h topical KCl application in intact or gonadectomized female and male rats, without or with estradiol or progesterone supplementation via daily intraperitoneal injections. Estrogen or progesterone treatment followed by withdrawal was studied in a separate cohort. To take the first step towards identifying potential mechanisms, we studied glutamate and GABA receptor binding using autoradiography.
RESULTS
The CSD frequency in intact female rats was higher than intact male and ovariectomized rats. We did not detect a change in CSD frequency during different stages of the estrous cycle in intact females. Daily estrogen injections for three weeks did not change CSD frequency. However, one-week estrogen withdrawal after two weeks of treatment significantly increased CSD frequency compared with the vehicle group in gonadectomized females. The same protocol of estrogen treatment and withdrawal was ineffective in gonadectomized males. In contrast to estrogen, daily progesterone injections for three weeks elevated CSD susceptibility, and one-week withdrawal after two weeks of treatment partially normalized this effect. Autoradiography did not reveal significant changes in glutamate or GABA receptor binding density after estrogen treatment and withdrawal.
CONCLUSIONS
These data suggest that females are more susceptible to CSD, and sexual dimorphism is abrogated by gonadectomy. Moreover, estrogen withdrawal after prolonged daily treatment enhances CSD susceptibility. These findings may have implications for estrogen-withdrawal migraine, although the latter tends to be without aura.
Topics: Rats; Female; Male; Animals; Cortical Spreading Depression; Progesterone; Receptors, GABA-A; Estrogens; Migraine Disorders; Glutamates
PubMed: 37237336
DOI: 10.1186/s10194-023-01598-x -
Plant Physiology Apr 2020Understanding the distribution of elements in plants is important for researchers across a broad range of fields, including plant molecular biology, agronomy, plant... (Review)
Review
Understanding the distribution of elements in plants is important for researchers across a broad range of fields, including plant molecular biology, agronomy, plant physiology, plant nutrition, and ionomics. However, it is often challenging to evaluate the applicability of the wide range of techniques available, with each having its own strengths and limitations. Here, we compare scanning/transmission electron microscopy-based energy-dispersive x-ray spectroscopy, x-ray fluorescence microscopy, particle-induced x-ray emission, laser ablation inductively coupled plasma-mass spectrometry, nanoscale secondary ion mass spectroscopy, autoradiography, and confocal microscopy with fluorophores. For these various techniques, we compare their accessibility, their ability to analyze hydrated tissues (without sample preparation) and suitability for in vivo analyses, as well as examining their most important analytical merits, such as resolution, sensitivity, depth of analysis, and the range of elements that can be analyzed. We hope that this information will assist other researchers to select, access, and evaluate the approach that is most useful in their particular research program or application.
Topics: Mass Spectrometry; Microscopy, Confocal; Microscopy, Electron; Plants; Spectrometry, X-Ray Emission
PubMed: 31974126
DOI: 10.1104/pp.19.01306 -
Molecular Biology of the Cell Aug 2021With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation...
With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.
Topics: Autoradiography; Chromatography, Gel; Ethylene Glycol; Osmotic Pressure; Phosphorylation; Polyethylene Glycols; Protein Conformation; Protein Kinases; Protein Multimerization; Scattering, Small Angle; WNK Lysine-Deficient Protein Kinase 1; Water; X-Ray Diffraction
PubMed: 33689398
DOI: 10.1091/mbc.E20-01-0089 -
BioRxiv : the Preprint Server For... Sep 2023Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain...
Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain tissue with elevated tau burden, purified filaments, and determined the structure of second-generation high avidity PET ligand MK-6240 at 2.31 Å resolution, which bound at a 1:1 ratio within the cleft of tau paired-helical filament (PHF), engaging with glutamine 351, lysine K353, and isoleucine 360. This information elucidates the basis of MK-6240 PET in quantifying PHF deposits in AD and may facilitate the structure-based design of superior ligands against tau amyloids.
PubMed: 37790438
DOI: 10.1101/2023.09.22.558671 -
Biomedicines Sep 2023Neuroinflammation is a complex biological process that typically originates as a protective response in the brain. This inflammatory process is triggered by the release... (Review)
Review
Neuroinflammation is a complex biological process that typically originates as a protective response in the brain. This inflammatory process is triggered by the release of pro-inflammatory substances like cytokines, prostaglandins, and reactive oxygen and nitrogen species from stimulated endothelial and glial cells, including those with pro-inflammatory functions, in the outer regions. While neuronal inflammation is common in various central nervous system disorders, the specific inflammatory pathways linked with different immune-mediated cell types and the various factors influencing the blood-brain barrier significantly contribute to disease-specific characteristics. The endocannabinoid system consists of cannabinoid receptors, endogenous cannabinoids, and enzymes responsible for synthesizing and metabolizing endocannabinoids. The primary cannabinoid receptor is CB1, predominantly found in specific brain regions such as the brainstem, cerebellum, hippocampus, and cortex. The presence of CB2 receptors in certain brain components, like cultured cerebellar granular cells, Purkinje fibers, and microglia, as well as in the areas like the cerebral cortex, hippocampus, and cerebellum is also evidenced by immunoblotting assays, radioligand binding, and autoradiography studies. Both CB1 and CB2 cannabinoid receptors exhibit noteworthy physiological responses and possess diverse neuromodulatory capabilities. This review primarily aims to outline the distribution of CB1 and CB2 receptors across different brain regions and explore their potential roles in regulating neuroinflammatory processes.
PubMed: 37893016
DOI: 10.3390/biomedicines11102642 -
Nature Neuroscience Jul 2023Dynamics and functions of neural circuits depend on interactions mediated by receptors. Therefore, a comprehensive map of receptor organization across cortical regions...
Dynamics and functions of neural circuits depend on interactions mediated by receptors. Therefore, a comprehensive map of receptor organization across cortical regions is needed. In this study, we used in vitro receptor autoradiography to measure the density of 14 neurotransmitter receptor types in 109 areas of macaque cortex. We integrated the receptor data with anatomical, genetic and functional connectivity data into a common cortical space. We uncovered a principal gradient of receptor expression per neuron. This aligns with the cortical hierarchy from sensory cortex to higher cognitive areas. A second gradient, driven by serotonin 5-HT receptors, peaks in the anterior cingulate, default mode and salience networks. We found a similar pattern of 5-HT expression in the human brain. Thus, the macaque may be a promising translational model of serotonergic processing and disorders. The receptor gradients may enable rapid, reliable information processing in sensory cortical areas and slow, flexible integration in higher cognitive areas.
Topics: Aged; Animals; Female; Humans; Male; Rats; Autoradiography; Brain Mapping; Cerebral Cortex; Cognition; Dendritic Spines; Gyrus Cinguli; Macaca fascicularis; Rats, Inbred Lew; Receptor, Serotonin, 5-HT1A; Receptors, Cholinergic; Receptors, Dopamine; Receptors, Neurotransmitter; Serotonin; Species Specificity; Myelin Sheath
PubMed: 37336976
DOI: 10.1038/s41593-023-01351-2 -
ACS Medicinal Chemistry Letters Oct 2022The long-lived radionuclides tritium and carbon-14 have been used for many years in pharmaceutical research and development for making key efficacy and toxicological... (Review)
Review
The long-lived radionuclides tritium and carbon-14 have been used for many years in pharmaceutical research and development for making key efficacy and toxicological decisions. Early discovery utilizes radiolabels for compound selection through radioligand binding assays and autoradiography. In preclinical safety evaluation, the use of labeled compounds for adsorption, distribution, metabolism, and excretion studies is often preferred for the added detection sensitivity. As the drug substance proceeds to the clinic, human metabolism studies are reliant on the use of labeled materials to fulfill required regulatory applications.
PubMed: 36262403
DOI: 10.1021/acsmedchemlett.2c00281