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Despite the recent advances in understanding the mechanisms of olfaction, no tools are currently available to noninvasively identify loss of smell. Because of the substantial increase in patients presenting with coronavirus disease 2019-related loss of smell, the pandemic has highlighted the urgent need to develop quantitative methods. Methods: Our group investigated the use of a novel fluorescent probe named Tsp1a-IR800P as a tool to diagnose loss of smell. Tsp1a-IR800P targets sodium channel 1.7, which plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. Results: Intuitively, we have identified that conditions leading to loss of smell, including chronic inflammation and coronavirus disease 2019, correlate with the downregulation of sodium channel 1.7 expression in the olfactory epithelium, both at the transcript and at the protein levels. We demonstrated that lower Tsp1a-IR800P fluorescence emissions significantly correlate with loss of smell in live animals-thus representing a potential tool for its semiquantitative assessment. Currently available methods rely on delayed subjective behavioral studies. Conclusion: This method could aid in significantly improving preclinical and clinical studies by providing a way to objectively diagnose loss of smell and therefore aid the development of therapeutic interventions.
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Trastornos del Olfato , Trastornos del Olfato/diagnóstico por imagen , Animales , Humanos , Imagen Óptica/métodos , Olfato , Colorantes Fluorescentes/química , Ratones , COVID-19/diagnóstico por imagen , MasculinoRESUMEN
Macrophages are key inflammatory mediators in many pathological conditions, including cardiovascular disease (CVD) and cancer, the leading causes of morbidity and mortality worldwide. This makes macrophage burden a valuable diagnostic marker and several strategies to monitor these cells have been reported. However, such strategies are often high-priced, non-specific, invasive, and/or not quantitative. Here, we developed a positron emission tomography (PET) radiotracer based on apolipoprotein A1 (ApoA1), the main protein component of high-density lipoprotein (HDL), which has an inherent affinity for macrophages. We radiolabeled an ApoA1-mimetic peptide (mA1) with zirconium-89 (89Zr) to generate a lipoprotein-avid PET probe (89Zr-mA1). We first characterized 89Zr-mA1's affinity for lipoproteins in vitro by size exclusion chromatography. To study 89Zr-mA1's in vivo behavior and interaction with endogenous lipoproteins, we performed extensive studies in wildtype C57BL/6 and Apoe-/- hypercholesterolemic mice. Subsequently, we used in vivo PET imaging to study macrophages in melanoma and myocardial infarction using mouse models. The tracer's cell specificity was assessed by histology and mass cytometry (CyTOF). Our data show that 89Zr-mA1 associates with lipoproteins in vitro. This is in line with our in vivo experiments, in which we observed longer 89Zr-mA1 circulation times in hypercholesterolemic mice compared to C57BL/6 controls. 89Zr-mA1 displayed a tissue distribution profile similar to ApoA1 and HDL, with high kidney and liver uptake as well as substantial signal in the bone marrow and spleen. The tracer also accumulated in tumors of melanoma-bearing mice and in the ischemic myocardium of infarcted animals. In these sites, CyTOF analyses revealed that natZr-mA1 was predominantly taken up by macrophages. Our results demonstrate that 89Zr-mA1 associates with lipoproteins and hence accumulates in macrophages in vivo. 89Zr-mA1's high uptake in these cells makes it a promising radiotracer for non-invasively and quantitatively studying conditions characterized by marked changes in macrophage burden.
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Surgeries and trauma result in traumatic and iatrogenic nerve damage that can result in a debilitating condition that approximately affects 189 million individuals worldwide. The risk of nerve injury during oncologic surgery is increased due to tumors displacing normal nerve location, blood turbidity, and past surgical procedures, which complicate even an experienced surgeon's ability to precisely locate vital nerves. Unfortunately, there is a glaring absence of contrast agents to assist surgeons in safeguarding vital nerves. To address this unmet clinical need, we leveraged the abundant expression of the voltage-gated sodium channel 1.7 (NaV1.7) as an intraoperative marker to access peripheral nerves in vivo, and visualized nerves for surgical guidance using a fluorescently-tagged version of a potent NaV1.7-targeted peptide, Tsp1a, derived from a Peruvian tarantula. We characterized the expression of NaV1.7 in sensory and motor peripheral nerves across mouse, primate, and human specimens and demonstrated universal expression. We synthesized and characterized a total of 10 fluorescently labeled Tsp1a-peptide conjugates to delineate nerves. We tested the ability of these peptide-conjugates to specifically accumulate in mouse nerves with a high signal-to-noise ratio in vivo. Using the best-performing candidate, Tsp1a-IR800, we performed thyroidectomies in non-human primates and demonstrated successful demarcation of the recurrent laryngeal and vagus nerves, which are commonly subjected to irreversible damage. The ability of Tsp1a to enhance nerve contrast during surgery provides opportunities to minimize nerve damage and revolutionize standards of care across various surgical specialties.
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In the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitudinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging approaches to investigate the immune response following myocardial infarction. The myocardial infarction models encompassed either transient or permanent left anterior descending coronary artery occlusion in C57BL/6 and Apoe-/-mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor 2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent occlusion model. Further, using 18F-fluorothymidine and 18F-fluorodeoxyglucose PET, we detected increased hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed persistent systemic inflammation and exacerbated atherosclerosis in Apoe-/- mice, regardless of which infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric imaging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical readouts.
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Introduction: Scanned fibre endomicroscopes are full point-scanning confocal microscopes with submicron lateral resolution with an optical slice thickness thin enough to isolate individual cell layers, allow active positioning of the optical slice in the z-axis and collection of megapixel images. Here we present descriptive findings and a brief atlas of an acquisition and annotation protocol high resolution in vivo capture of oral mucosal pathology including oral squamous cell carcinoma and dysplasia using a fluorescence scanned fibre endomicroscope with 3 topical fluorescent imaging agents: fluorescein, acriflavine and PARPi-FL. Methods: Digital biopsy was successfully performed via an acquisition protocol in seventy-one patients presenting for investigation of oral mucosal abnormalities using a miniaturized, handheld scanned fibre endoscope. Multiple imaging agents were utilized and multiple time points sampled. Fifty-nine patients had a matched histopathology correlating in location with imaging. The images were annotated back to macrographic location using a purpose-built software, MouthMap™. Results: Acquisition and annotation of cellular level resolved images was demonstrated with all 3 topical agents. Descriptive observations between clinically or histologically normal oral mucosa showed regular intranuclear distance, a regular nuclear profile and fluorescent homogeneity. This was dependent on the intraoral location and type of epithelium being observed. Key features of malignancy were a loss of intranuclear distance, disordered nuclear clustering and irregular nuclear fluorescence intensity and size. Perinuclear fluorescent granules were seen in the absence of irregular nuclear features in lichenoid inflammation. Discussion: High resolution oral biopsy allows for painless and rapid capture of multiple mucosal sites, resulting in more data points to increase diagnostic precision. High resolution digital micrographs can be easily compared serially across multiple time points utilizing an annotation software. In the present study we have demonstrated realization of a high-resolution digital biopsy protocol of the oral mucosa for utility in the diagnosis of oral cancer and precancer..
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We report an innovative approach to producing bacteriochlorins (bacs) via formal cycloaddition by subjecting a porphyrin to a trimolecular reaction. Bacs are near-infrared probes with the intrinsic ability to serve in multimodal imaging. However, despite their ability to fluoresce and chelate metal ions, existing bacs have thus offered limited ability to label biomolecules for target specificity or have lacked chemical purity, limiting their use in bio-imaging. In this work, bacs allowed a precise and controlled appending of clickable linkers, lending the porphyrinoids substantially more chemical stability, clickability, and solubility, rendering them more suitable for preclinical investigation. Our bac probes enable the targeted use of biomolecules in fluorescence imaging and Cerenkov luminescence for guided intraoperative imaging. Bacs' capacity for chelation provides opportunities for use in non-invasive positron emission tomography/computed tomography. Herein, we report the labeling of bacs with Hs1a, a (NaV1.7)-sodium-channel-binding peptide derived from the Chinese tarantula Cyriopagopus schmidti to yield Bac-Hs1a and radiolabeled Hs1a, which shuttles our bac sensor(s) to mouse nerves. In vivo, the bac sensor allowed us to observe high signal-to-background ratios in the nerves of animals injected with fluorescent Bac-Hs1a and radiolabeled Hs1a in all imaging modes. This study demonstrates that Bac-Hs1a and [64Cu]Cu-Bac-Hs1a accumulate in peripheral nerves, providing contrast and utility in the preclinical space. For the chemistry and bio-imaging fields, this study represents an exciting starting point for the modular manipulation of bacs, their development and use as probes for diagnosis, and their deployment as formidable multiplex nerve-imaging agents for use in routine imaging experiments.
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Porfirinas , Animales , RatonesRESUMEN
PARPi-FL is a molecularly specific fluorescent agent that targets poly ADP-ribose polymerase 1, a DNA repair enzyme overexpressed in the nuclei of tumor cells. This imaging agent is being investigated in a clinical trial (NCT03085147) for the detection of oral cancer. The PARPi-FL mouthwash formulation currently being used in the phase I/II clinical trial comprises 1,000 nM of PARPi-FL dissolved first in 4.5 ml of polyethylene glycol (PEG) 300 and then in 9.5 ml of water. This formulation requires a 2-step process that can be cumbersome for routine clinical use. To minimize errors and simplify the formulation process, we have developed a new one-step formulation, which requires only the direct addition of water into a vial containing a mixture of the PARPi-FL and PEG 3350, which is also a powder. In a series of analytical and preclinical studies, we demonstrate that the new formulation of PARPi-FL is stable over 365 days, sustains its characteristics, and performs similar to the previous formulation. Moving forward, the new formulation of the PARPi-FL will be used for patients accrued in the phase II clinical trial.
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Neoplasias de la Boca , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , PolietilenglicolesRESUMEN
PURPOSE: 123I-MAPi, a novel PARP1-targeted Auger radiotherapeutic has shown promising results in pre-clinical glioma model. Currently, 123I-MAPi is synthesized using multistep synthesis that results in modest yields and low molar activities (MA) that limits the ability to translate this technology for human studies where high doses are administered. Therefore, new methods are needed to synthesize 123I-MAPi in high activity yields (AY) and improved MA to facilitate clinical translation and multicenter trials. MATERIALS AND METHODS: 123I-MAPi was prepared in a single step via 123I-iododetannylation of the corresponding tributylstannane precursor. In vitro internalization assay, subcellular fractionation and confocal microscopy where used to evaluate the performance of 123I-MAPi in a small cell lung cancer model. RESULTS: 123I-MAPi was synthesized in a single step from the corresponding stannane precursor in AY of 45 ± 2% and MA of 11.8 ± 4.8 GBq µmol-1. In vitro in LX22 cells showed rapid internalization (5 min) with accumulation found predominantly in the membrane, nucleus and chromatin of the cell as determined by subcellular fractionation. CONCLUSIONS: Here, we have developed an improved radiosynthesis of 123I-MAPi, an Auger theranostic agent. This process was achieved using a single step, 123I-iododestannylation reaction from the corresponding stannane precursor in good AY and MA. 123I-MAPi was evaluated in vitro in a small cell lung cancer model with high PARP expression, rapid internalization and high nuclear uptake shown.
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Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Humanos , Medicina de Precisión , ElectronesRESUMEN
The sense of smell (olfaction) is one of the most important senses for animals including humans. Despite significant advances in the understanding mechanism of olfaction, currently, there are no objective non-invasive methods that can identify loss of smell. Covid-19-related loss of smell has highlighted the need to develop methods that can identify loss of olfaction. Voltage-gated sodium channel 1.7 (NaV1.7) plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. We have identified several conditions such as chronic inflammation and viral infections such as Covid-19 that lead to loss of smell correlate with downregulation of NaV1.7 expression at transcript and protein levels in the olfactory epithelium. Leveraging this knowledge, we have developed a novel fluorescent probe Tsp1a-IR800 that targets NaV1.7. Using fluorescence imaging we can objectively measure the loss of sense of smell in live animals non-invasively. We also demonstrate that our non-invasive method is semiquantitative because the loss of fluorescence intensity correlates with the level of smell loss. Our results indicate, that our probe Tsp1a-IR800, can objectively diagnose anosmia in animal and human subjects using infrared fluorescence. We believe this method to non-invasively diagnose loss of smell objectively is a significant advancement in relation to current methods that rely on highly subjective behavioral studies and can aid in studying olfaction loss and the development of therapeutic interventions.
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In recent years, cardiovascular immuno-imaging by positron emission tomography (PET) has undergone tremendous progress in preclinical settings. Clinically, two approved PET tracers hold great potential for inflammation imaging in cardiovascular patients, namely FDG and DOTATATE. While the former is a widely applied metabolic tracer, DOTATATE is a relatively new PET tracer targeting the somatostatin receptor 2 (SST2). In the current study, we performed a detailed, head-to-head comparison of DOTATATE-based radiotracers and [18F]F-FDG in mouse and rabbit models of cardiovascular inflammation. For mouse experiments, we labeled DOTATATE with the long-lived isotope [64Cu]Cu to enable studying the tracer's mode of action by complementing in vivo PET/CT experiments with thorough ex vivo immunological analyses. For translational PET/MRI rabbit studies, we employed the more widely clinically used [68Ga]Ga-labeled DOTATATE, which was approved by the FDA in 2016. DOTATATE's pharmacokinetics and timed biodistribution were determined in control and atherosclerotic mice and rabbits by ex vivo gamma counting of blood and organs. Additionally, we performed in vivo PET/CT experiments in mice with atherosclerosis, mice subjected to myocardial infarction and control animals, using both [64Cu]Cu-DOTATATE and [18F]F-FDG. To evaluate differences in the tracers' cellular specificity, we performed ensuing ex vivo flow cytometry and gamma counting. In mice subjected to myocardial infarction, in vivo [64Cu]Cu-DOTATATE PET showed higher differential uptake between infarcted (SUVmax 1.3, IQR, 1.2-1.4, N = 4) and remote myocardium (SUVmax 0.7, IQR, 0.5-0.8, N = 4, p = 0.0286), and with respect to controls (SUVmax 0.6, IQR, 0.5-0.7, N = 4, p = 0.0286), than [18F]F-FDG PET. In atherosclerotic mice, [64Cu]Cu-DOTATATE PET aortic signal, but not [18F]F-FDG PET, was higher compared to controls (SUVmax 1.1, IQR, 0.9-1.3 and 0.5, IQR, 0.5-0.6, respectively, N = 4, p = 0.0286). In both models, [64Cu]Cu-DOTATATE demonstrated preferential accumulation in macrophages with respect to other myeloid cells, while [18F]F-FDG was taken up by macrophages and other leukocytes. In a translational PET/MRI study in atherosclerotic rabbits, we then compared [68Ga]Ga-DOTATATE and [18F]F-FDG for the assessment of aortic inflammation, combined with ex vivo radiometric assays and near-infrared imaging of macrophage burden. Rabbit experiments showed significantly higher aortic accumulation of both [68Ga]Ga-DOTATATE and [18F]F-FDG in atherosclerotic (SUVmax 0.415, IQR, 0.338-0.499, N = 32 and 0.446, IQR, 0.387-0.536, N = 27, respectively) compared to control animals (SUVmax 0.253, IQR, 0.197-0.285, p = 0.0002, N = 10 and 0.349, IQR, 0.299-0.423, p = 0.0159, N = 11, respectively). In conclusion, we present a detailed, head-to-head comparison of the novel SST2-specific tracer DOTATATE and the validated metabolic tracer [18F]F-FDG for the evaluation of inflammation in small animal models of cardiovascular disease. Our results support further investigations on the use of DOTATATE to assess cardiovascular inflammation as a complementary readout to the widely used [18F]F-FDG.
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Aterosclerosis , Infarto del Miocardio , Compuestos Organometálicos , Animales , Aterosclerosis/diagnóstico por imagen , Fluorodesoxiglucosa F18/metabolismo , Radioisótopos de Galio , Humanos , Inflamación/diagnóstico por imagen , Ratones , Infarto del Miocardio/diagnóstico por imagen , Compuestos Organometálicos/metabolismo , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tomografía de Emisión de Positrones/métodos , Conejos , Cintigrafía , Radiofármacos , Distribución TisularRESUMEN
Indolo[2,3-d]benzazepines (indololatonduines) are rarely discussed in the literature. In this project, we prepared a series of novel indololatonduine derivatives and their RuII and OsII complexes and investigated their microtubule-targeting properties in comparison with paclitaxel and colchicine. Compounds were fully characterized by spectroscopic techniques (1H NMR and UV-vis), ESI mass-spectrometry, and X-ray crystallography, and their purity was confirmed by elemental analysis. The stabilities of the compounds in DMSO and water were confirmed by 1H and 13C NMR and UV-vis spectroscopy. Novel indololatonduines demonstrated anticancer activity in vitro in a low micromolar concentration range, while their coordination to metal centers resulted in a decrease of cytotoxicity. The preliminary in vivo activity of the RuII complex was investigated. Fluorescence staining and in vitro tubulin polymerization assays revealed the prepared compounds to have excellent microtubule-destabilizing activities, even more potent than the well-known microtubule-destabilizing agent colchicine.
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Antineoplásicos/farmacología , Complejos de Coordinación/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Indoles/farmacología , Microtúbulos/efectos de los fármacos , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Heterocíclicos con 3 Anillos/química , Humanos , Indoles/química , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Microscopía Fluorescente , Microtúbulos/metabolismo , Modelos Moleculares , Estructura Molecular , Polimerizacion/efectos de los fármacos , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
OBJECTIVE: The aim of this study was to develop and validate a pretreatment prognostic score in pancreatic cancer (PDAC). BACKGROUND: Pretreatment prognostication in PDAC is important for treatment decisions but remains challenging. Available prognostic tools are derived from selected cohorts of patients who underwent resection, excluding up to 20% of patients with exploration only, and do not adequately reflect the pretreatment scenario. METHODS: Patients undergoing surgery for PDAC in Heidelberg from July 2006 to June 2014 were identified from a prospective database. Pretreatment parameters were extracted from the database and the laboratory information system. Parameters independently associated with overall survival by uni- and multivariable analyses were used to build a prognostic score. A contemporary cohort from Verona was used for external validation. RESULTS: In 1197 patients, multiple pretreatment parameters were associated with overall survival by univariable analyses. American Society of Anesthesiology classification, carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen, C-reactive protein, albumin, and platelet count were independently associated with survival and were used to create the Heidelberg Prognostic Pancreatic Cancer (HELPP)-score. The HELPP-score was closely associated with overall survival (median survival between 31.3 and 4.8 months; 5-year survival rates between 35% and 0%) and was able to stratify survival in subgroups with or without resection as well as in CA19-9 nonsecretors. In the resected subgroup the HELPP-score stratified survival independently of pathological prognostic factors. The HELPP-score was externally validated and was superior to CA19-9 in both the development and validation cohorts. CONCLUSION: The HELPP-score is a readily available prognostic tool based on pretreatment routine parameters to stratify survival in PDAC independently of resection status and pathological tumor stage.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Antígeno CA-19-9 , Pronóstico , Neoplasias Pancreáticas/patología , Albúminas , Carcinoma Ductal Pancreático/patología , Estudios Retrospectivos , Neoplasias PancreáticasRESUMEN
Reflectance confocal microscopy (RCM) with endogenous backscattered contrast can noninvasively image basal cell carcinomas (BCCs) in skin. However, BCCs present with high nuclear density, and the relatively weak backscattering from nuclei imposes a fundamental limit on contrast, detectability, and diagnostic accuracy. We investigated PARPi-FL, an exogenous nuclear poly(adenosine diphosphate ribose) polymerase (PARP1)-targeted fluorescent contrast agent, and fluorescence confocal microscopy toward improving BCC diagnosis. Methods: We tested PARP1 expression in 95 BCC tissues using immunohistochemistry, followed by PARPi-FL staining in 32 fresh surgical BCC specimens. The diagnostic accuracy of PARPi-FL contrast was evaluated in 83 surgical specimens. The optimal parameters for permeability of PARPi-FL through intact skin was tested ex vivo on 5 human skin specimens and in vivo in 3 adult Yorkshire pigs. Results: We found significantly higher PARP1 expression and PARPi-FL binding in BCCs than in normal skin structures. Blinded reading of RCM-and-fluorescence confocal microscopy images by 2 experts demonstrated a higher diagnostic accuracy for BCCs with combined fluorescence and reflectance contrast than for RCM alone. Optimal parameters (time and concentration) for PARPi-FL transepidermal permeation through intact skin were successfully determined. Conclusion: Combined fluorescence and reflectance contrast may improve noninvasive BCC diagnosis with confocal microscopy.
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Carcinoma Basocelular , Neoplasias Cutáneas , Animales , Carcinoma Basocelular/diagnóstico por imagen , Carcinoma Basocelular/patología , Carcinoma Basocelular/cirugía , Núcleo Celular/patología , Inmunohistoquímica , Microscopía Confocal/métodos , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/patología , PorcinosRESUMEN
The human nociceptor-specific voltage-gated sodium channel 1.7 (hNaV1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNaV1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNaV1.7 (IC50 = 10 nM), with greater than 100-fold selectivity over hNaV1.3-hNaV1.6, 45-fold selectivity over hNaV1.1, and 24-fold selectivity over hNaV1.2. Tsp1a is a gating modifier that inhibits NaV1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNaV1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral pain.
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Background: Human papillomavirus- (HPV-) associated oropharyngeal squamous cell carcinomas (OPSCCs) are clinically and pathologically distinct from HPV-negative tumors. Here, we explore whether HPV affects functional biomarkers, including γH2AX, RAD51, and PARP1. Moreover, the role of [18F]PARPi as a broadly applicable imaging tool for head and neck carcinomas is investigated. Methods: HPV-positive and HPV-negative cell lines were used to evaluate the γH2AX, RAD51, and PARP1 expression with immunoblotting and immunofluorescence. Effects of external beam ionizing radiation were investigated in vitro, and survival was investigated via colony-formation assay. [18F]PARPi uptake experiments were performed on HPV-negative and HPV-positive cell lines to quantify PARP1 expression. PARP1 IHC and γH2AX foci were quantified using patient-derived oropharyngeal tumor specimens. Results: Differences in DNA repair were detected, showing higher RAD51 and γH2AX expression in HPV-positive cell lines. Clonogenic assays confirm HPV-positive cell lines to be significantly more radiosensitive. PARP1 expression levels were similar, irrespective of HPV status. Consequently, [18F]PARPi uptake assays demonstrated that this tracer is internalized in cell lines independently from their HPV status. Conclusion: The HPV status, often used clinically to stratify patients, did not affect PARP1 levels, suggesting that PARP imaging can be performed in both HPV-positive and HPV-negative patients. This study confirms that the PET imaging agent [18F]PARPi could serve as a general clinical tool for oropharyngeal cancer patients.
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Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Humanos , Neoplasias Orofaríngeas/diagnóstico por imagen , Papillomaviridae , Infecciones por Papillomavirus/diagnóstico por imagenRESUMEN
Despite Auger electrons being highly appealing due to their short-range and high linear energy transfer to surrounding tissues, the progress in the field has been limited due to the challenge in delivering a therapeutic dose within the close proximity of cancer cell's DNA. Here, we demonstrate that the PARP inhibitor 123I-MAPi is a viable agent for the systemic administration and treatment of p53 mutant cancers. Significantly, minimal off-site toxicity was observed in mice administered with up to 74 MBq of 127I-PARPi. Taken together, these results lay the foundation for future clinical evaluation and broader preclinical investigations. By harnessing the scaffold of the PARP inhibitor Olaparib, we were able to deliver therapeutic levels of Auger radiation to the site of human colorectal cancer xenograft tumors after systemic administration. In-depth toxicity studies analyzed blood chemistry levels and markers associated with specific organ toxicity. Finally, p53+/+ and p53-/- human colorectal cancer cell lines were evaluated for the ability of 123I-MAPi to induce tumor growth delay. Toxicity studies demonstrate that both 123I-MAPi and its stable isotopologue, 127I-PARPi, have no significant off-site toxicity when administered systemically. Analysis following 123I-MAPi treatment confirmed its ability to induce DNA damage at the site of xenograft tumors when administered systemically. Finally, we demonstrate that 123I-MAPi generates a therapeutic response in p53-/-, but not p53+/+, subcutaneous xenograft tumors in mouse models. Taken together, these results represent the first example of a PARP Auger theranostic agent capable of delivering a therapeutic dose to xenograft human colorectal cancer tumors upon systemic administration without causing significant toxicity to surrounding mouse organs. Moreover, it suggests that a PARP Auger theranostic can act as a targeted therapeutic for cancers with mutated p53 pathways. This landmark goal paves the way for clinical evaluation of 123I-MAPi for pan cancer therapeutics.
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Quimioradioterapia/métodos , Neoplasias del Colon/terapia , Electrones/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Nanomedicina Teranóstica/métodos , Animales , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Femenino , Humanos , Ratones , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recruitment and activation of the ataxia telangiectasia mutated (ATM) kinase regulate multiple cell-cycle checkpoints relevant to complex biological events like DNA damage repair and apoptosis. Molecularly specific readouts of ATM using protein assays, fluorescence, or radiolabeling have advanced significantly over the past few years. This Review covers the molecular imaging techniques that enable the visualization of ATM-from traditional quantitative protein assays to the potential use of ATM inhibitors to generate new imaging agents to interrogate ATM. We are confident that molecular imaging coupled with advanced technologies will play a pivotal role in visualizing and understanding the biology of ATM and accelerate its applications in the diagnosis and monitoring of disease, including radiation therapy and patient stratification.
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Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ataxia Telangiectasia/diagnóstico , Imagen Molecular/métodos , Inhibidores de Proteínas Quinasas/farmacología , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/terapia , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , HumanosRESUMEN
BACKGROUND: Visual inspection and biopsy is the current standard of care for oral cancer diagnosis, but is subject to misinterpretation and consequently to misdiagnosis. Topically applied PARPi-FL is a molecularly specific, fluorescent contrast-based approach that may fulfill the unmet need for a simple, in vivo, non-invasive, cost-effective, point-of-care method for the early diagnosis of oral cancer. Here, we present results from a phase I safety and feasibility study on fluorescent, topically applied PARPi-FL. Twelve patients with a histologically proven oral squamous cell carcinoma (OSCC) gargled a PARPi-FL solution for 60 s (15 mL, 100 nM, 250 nM, 500 nM, or 1000 nM), followed by gargling a clearing solution for 60 s. Fluorescence measurements of the lesion and surrounding oral mucosa were taken before PARPi-FL application, after PARPi-FL application, and after clearing. Blood pressure, oxygen levels, clinical chemistry, and CBC were obtained before and after tracer administration. RESULTS: PARPi-FL was well-tolerated by all patients without any safety concerns. When analyzing the fluorescence signal, all malignant lesions showed a significant differential in contrast after administration of PARPi-FL, with the highest increase occurring at the highest dose level (1000 nM), where all patients had a tumor-to-margin fluorescence signal ratio of >3. A clearing step was essential to increase signal specificity, as it clears unbound PARPi-FL trapped in normal anatomical structures. PARPi-FL tumor cell specificity was confirmed by ex vivo tabletop confocal microscopy. We have demonstrated that the fluorescence signal arose from the nuclei of tumor cells, endorsing our macroscopic findings. CONCLUSIONS: A PARPi-FL swish & spit solution is a rapid and non-invasive diagnostic tool that preferentially localizes fluorescent contrast to OSCC. This technique holds promise for the early detection of OSCC based on in vivo optical evaluation and targeted biopsy of suspicious lesions in the oral cavity. TRIAL REGISTRATION: Clinicaltrials.gov -NCT03085147, registered on March 21st, 2017.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de la Boca , Carcinoma de Células Escamosas/diagnóstico por imagen , Colorantes Fluorescentes , Humanos , Neoplasias de la Boca/diagnóstico por imagen , Poli(ADP-Ribosa) Polimerasa-1RESUMEN
PURPOSE: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus 2019 disease (COVID-19), poses a serious risk to humanity and represents a huge challenge for healthcare systems worldwide. Since the early days of the COVID-19 pandemic, it has been evident that adequate testing is an essential step in limiting and controlling the spread of SARS-CoV-2. Here, we present an accurate, inexpensive, scalable, portable, and rapid detection kit to directly detect SARS-CoV-2 in biological samples that could even be translated for population testing. We have demonstrated that our method can reliably identify viral load and could be used to reach those fractions of the population with limited access to more sophisticated and expensive tests. PROCEDURES: The proposed SARS-CoV-2 detection kit is based on the combination of a SARS-CoV-2-targeted antibody (CR3022) that targets spike protein S1 domain on the viral surface. This antibody was radiolabeled with a long-lived isotope (Iodine-125) to allow us to detect bound antibody in samples with SARS-CoV-2. We used a series of in vitro assays to determine sensitivity and specificity and facilitate automation of the testing kit. Bound antibody was extracted from saliva samples via a centrifugation step and a semi-permeable membrane. Our kit was further validated using SARS-CoV-2 virions. RESULTS: We were able to accomplish radiosynthesis of [125I]I-CR3022 reliably without loss of binding. The SARS-CoV-2-sensing antibody was shown to maintain its spike S1 affinity and to bind to as low as 2.5-5 ng of spike protein. We then used beads-bound spike S1 to develop a separation kit which proved to be both easy to use and inexpensive. The kit made it possible to extract bound antibody from the saliva-like sample. We were able to validate the separation kit using intact SARS-CoV-2 virions and showed that our kit can detect a viral concentration as low as 19,700 PFU/mL (~ 9.22%TBF) and as high as 1,970,000 PFU/mL (45.04%TBF). CONCLUSION: Here we report the development and validation of a SARS-CoV-2 detection system based on the combination of a specific radiolabeled antibody and a separation membrane. We demonstrate our system to be comparable to other SARS-CoV-2 detection kits already approved by the FDA and believe this technology could be easily deployed to countries with limited resources for the diagnosis of COVID-19. Furthermore, workflows could be easily adapted to target other antigens and therefore other types of diseases.