Neural substrates of cough control during coughing.

Cough is known as a protective reflex to keep the airway free from harmful substances. Although brain activity during cough was previously examined mainly by functional magnetic resonance imaging (fMRI) with model analysis, this method does not capture real brain activity during cough. To obtain accurate measurements of brain activity during cough, we conducted whole-brain scans during different coughing tasks while correcting for head motion using a restraint-free positron emission tomography (PET) system. Twenty-four healthy right-handed males underwent multiple PET scans with [15O]H2O. Four tasks were performed during scans: "resting"; "voluntary cough (VC)", which simply repeated spontaneous coughing; "induced cough (IC)", where participants coughed in response to an acid stimulus in the cough-inducing method with tartaric acid (CiTA); and "suppressed cough (SC)", where coughing was suppressed against CiTA. The whole brain analyses of motion-corrected data revealed that VC chiefly activated the cerebellum extending to pons. In contrast, CiTA-related tasks (IC and SC) activated the higher sensory regions of the cerebral cortex and associated brain regions. The present results suggest that brain activity during simple cough is controlled chiefly by infratentorial areas, whereas manipulating cough predominantly requires the higher sensory brain regions to allow top-down control of information from the periphery.

Effective nose-to-brain drug delivery using a combination system targeting the olfactory region in monkeys.

The nose-to-brain (N2B) pathway has garnered attention because it transports drugs directly into the brain. Although recent studies have suggested the necessity of selective drug administration to the olfactory region for effective N2B drug delivery, the importance of delivering the formulation to the olfactory region and the detailed pathway involved in drug uptake in primates brain remain unclear. Here, we developed a combination system for N2B drug delivery comprising a proprietary mucoadhesive powder formulation and a dedicated nasal device (N2B-system) and evaluated it for nasal drug delivery to the brain in cynomolgus monkeys. This N2B-system demonstrated a much greater formulation distribution ratio in the olfactory region in an in vitro experiment using a 3D-printed nasal cast and in vivo experiment using cynomolgus monkeys, as compared to that in other nasal drug delivery systems that comprise of a proprietary nasal powder device developed for nasal absorption and vaccination and a commercially available liquid spray. Additionally, Texas Red-labeled dextran (TR-DEX, 3 kDa) was administered using the N2B-system to estimate the drug transition pathway from the nasal cavity to the brain. TR-DEX preferentially localized to the olfactory epithelium and reached the olfactory bulb through the cribriform foramina. Moreover, domperidone, a model drug with poor blood-brain barrier permeability, was administered to assess the brain uptake of medicine after olfactory region-selective administration by using the N2B-system. Domperidone accumulation in the brain was evaluated using positron emission tomography with intravenously administered [18F]fallypride based on competitive inhibition of the dopamine D2 receptor (D2R). Compared to other systems, the N2B-system significantly increased D2R occupancy and domperidone uptake in the D2R-expressing brain regions. The current study reveals that the olfactory region of the nasal cavity is a suitable target for efficient nasal drug delivery to the brain in cynomolgus monkeys. Thus, the N2B-system, which targets the olfactory region, provides an efficient approach for developing effective technology for nasal drug delivery to the brain in humans.

In vivo imaging of acute physiological responses after treatment of cancer with near-infrared photoimmunotherapy.

PURPOSE: Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer phototherapy using an antibody-photosensitizer conjugate (Ab-IR700). By NIR light irradiation, Ab-IR700 forms a water-insoluble aggregation on the plasma membrane of cancer cells, leading to lethal membrane damage of cancer cells with high selectivity. However, IR700 produces singlet oxygen, which induces non-selective inflammatory responses such as edema in normal tissues around the tumor. Understanding such treatment-emergent responses is important to minimize side effects and improve clinical outcomes. Thus, in this study, we evaluated physiological responses during NIR-PIT by magnetic resonance imaging (MRI) and positron emission tomography (PET). PROCEDURES: Ab-IR700 was intravenously injected into tumor-bearing mice with two tumors on the right and left sides of the dorsum. At 24 h after injection, a tumor was irradiated with NIR light. Edema formation was examined by T1/T2/diffusion-weighted MRI and inflammation was investigated by PET with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). Because inflammation can increase vascular permeability via inflammatory mediators, we evaluated changes in oxygen levels in tumors using a hypoxia imaging probe, [18F]fluoromisonidazole ([18F]FMISO). RESULTS: The uptake of [18F]FDG in the irradiated tumor was significantly decreased compared to the control tumor, indicating the impairment of glucose metabolism induced by NIR-PIT. MRI and [18F]FDG-PET images showed that inflammatory edema with [18F]FDG accumulation was present in the surrounding normal tissues of the irradiated tumor. Furthermore, [18F]FMISO accumulation in the center of the irradiated tumor was relatively low, indicating the enhancement of oxygen supply due to increased vascular permeability. In contrast, high [18F]FMISO accumulation was observed in the peripheral region, indicating enhancement of hypoxia in the region. This could be because inflammatory edema was formed in the surrounding normal tissues, which blocked blood flow to the tumor. CONCLUSIONS: We successfully monitored inflammatory edema and changes in oxygen levels during NIR-PIT. Our findings on the acute physiological responses after light irradiation will help to develop effective measures to minimize the side effects in NIR-PIT.

Reduced P-glycoprotein recognition of a radioiodine-labeled phosphonium cation by stilbenylation for mitochondrial membrane potential based-imaging.

The accumulation of radiolabeled phosphonium cations in cells is dependent on the mitochondrial membrane potential (MMP). However, the efflux of these cations from tumor cells via P-glycoprotein (P-gp) limits their clinical application as MMP-based imaging tracers. In the present study, we designed (E)-diethyl-4-[125I]iodobenzyl-4-stilbenylphosphonium ([125I]IDESP), which contains a stilbenyl substituent, as a P-gp inhibitor to reduce P-gp recognition, and evaluated its biological properties in comparison with 4-[125I]iodobenzyl dipropylphenylphosphonium ([125I]IDPP). The in vitro cellular uptake ratio of [125I]IDESP in P-gp expressing K562/Vin cells to the parent (P-gp negative) K562 cells was significantly higher than that of [125I]IDPP. The efflux rate of [125I]IDESP was not significantly different between K562 and K562/Vin, while [125I]IDPP was rapidly effluxed from K562/Vin compared with K562, and the efflux of [125I]IDPP from K562/Vin was inhibited by the P-gp inhibitor, cyclosporine A. The cellular uptake of [125I]IDESP was well correlated with the MMP levels. These results suggested that [125I]IDESP was accumulated in cells depending on the MMP levels, without being effluxed via P-gp, while [125I]IDPP was rapidly effluxed from the cells via P-gp. Despite having suitable in vitro properties for MMP-based imaging, [125I]IDESP showed rapid blood clearance and lower tumor accumulation than [125I]IDPP. Improvement in the normal tissue distribution of [125I]IDESP is required to develop an agent for use in in vivo MMP-based tumor imaging.

Elucidation of the Role of FAM210B in Mitochondrial Metabolism and Erythropoiesis.

Mitochondria play essential and specific roles during erythroid differentiation. Recently, FAM210B, encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD+)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.

Measurement of Hepatic CYP3A4 and 2D6 Activity Using Radioiodine-Labeled O-Desmethylvenlafaxine.

Drug metabolizing enzyme activity is affected by various factors such as drug-drug interactions, and a method to quantify drug metabolizing enzyme activity in real time is needed. In this study, we developed a novel radiopharmaceutical for quantitative imaging to estimate hepatic CYP3A4 and CYP2D6 activity. Iodine-123- and 125-labeled O-desmethylvenlafaxine (123/125I-ODV) was obtained with high labeling and purity, and its metabolism was found to strongly involve CYP3A4 and CYP2D6. SPECT imaging in normal mice showed that the administered 123I-ODV accumulated early in the liver and was excreted into the gallbladder, as evaluated by time activity curves. In its biological distribution, 125I-ODV administered to mice accumulated early in the liver, and only the metabolite of 125I-ODV was quickly excreted into the bile. In CYP3A4- and CYP2D6-inhibited model mice, the accumulation in bile decreased more than in normal mice, indicating inhibition of metabolite production. These results indicated that imaging and quantifying the accumulation of radioactive metabolites in excretory organs will aid in determining the dosages of various drugs metabolized by CYP3A4 and CYP2D6 for individualized medicine. Thus, 123/125I-ODV has the potential to direct, comprehensive detection and measurement of hepatic CYP3A4 and CYP2D6 activity by a simple and less invasive approach.

Exploring the mechanistic link between SF3B1 mutation and ring sideroblast formation in myelodysplastic syndrome.

Acquired sideroblastic anemia, characterized by bone marrow ring sideroblasts (RS), is predominantly associated with myelodysplastic syndrome (MDS). Although somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery, are frequently found in MDS-RS, the detailed mechanism contributing to RS formation is unknown. To explore the mechanism, we established human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells stably expressing SF3B1K700E. SF3B1K700E expressing cells showed higher proportion of RS than the control cells along with erythroid differentiation, indicating the direct contribution of mutant SF3B1 expression in erythroblasts to RS formation. In SF3B1K700E expressing cells, ABCB7 and ALAS2, known causative genes for congenital sideroblastic anemia, were downregulated. Additionally, mis-splicing of ABCB7 was observed in SF3B1K700E expressing cells. ABCB7-knockdown HUDEP-2 cells revealed an increased frequency of RS formation along with erythroid differentiation, demonstrating the direct molecular link between ABCB7 defects and RS formation. ALAS2 protein levels were obviously decreased in ABCB7-knockdown cells, indicating decreased ALAS2 translation owing to impaired Fe-S cluster export by ABCB7 defects. Finally, RNA-seq analysis of MDS clinical samples demonstrated decreased expression of ABCB7 by the SF3B1 mutation. Our findings contribute to the elucidation of the complex mechanisms of RS formation in MDS-RS.

COVID-19 in a Hairy Cell Leukemia Patient: A Rare Case Report.

The detailed clinical course of coronavirus disease 2019 (COVID-19) in patients with hairy cell leukemia (HCL) is rarely reported. We report the first case of HCL diagnosed with prolonged pancytopenia after COVID-19 infection in Japan. We describe the case of a 56-year-old man who was diagnosed with COVID-19. Computed tomography revealed ground-glass opacities in the bilateral lung lobes as well as splenomegaly. Remdesivir and dexamethasone were administered for the treatment of COVID-19. Since the pancytopenia persisted, bone marrow examination was performed, and he was diagnosed with HCL. Although pancytopenia can occur with COVID-19 alone, clinicians should be alerted regarding the presence of hematologic malignancies in patients in whom pancytopenia persists after COVID-19 treatment or in those with splenomegaly. Further, the condition of all previously reported patients with COVID-19 associated with HCL was severe enough to require mechanical ventilation. This is the first case in which the disease was not severe. The interleukin-6 (IL-6) level was lower in this case than in previous cases, suggesting that racial differences in IL-6 production may have contributed to COVID-19 severity.

Indirect SPECT Imaging Evaluation for Possible Nose-to-Brain Drug Delivery Using a Compound with Poor Blood-Brain Barrier Permeability in Mice.

Single-photon emission computed tomography (SPECT) imaging using intravenous radioactive ligand administration to indirectly evaluate the time-dependent effect of intranasal drugs with poor blood-brain barrier permeability on brain drug distributions in mice was evaluated. The biodistribution was examined using domperidone, a dopamine D2 receptor ligand, as the model drug, with intranasal administration at 0, 15, or 30 min before intravenous [123I]IBZM administration. In the striatum, [123I]IBZM accumulation was significantly lower after intranasal (IN) domperidone administration than in controls 15 min after intravenous [125I]IBZM administration. [123I]IBZM SPECT was acquired with intravenous (IV) or IN domperidone administration 15 min before [123I]IBZM, and time-activity curves were obtained. In the striatum, [123I]IBZM accumulation was clearly lower in the IN group than in the control and IV groups. Time-activity curves showed no significant difference between the control and IV groups in the striatum, and values were significantly lowest during the first 10 min in the IN group. In the IN group, binding potential and % of receptor occupancy were significantly lower and higher, respectively, compared to the control and IV groups. Thus, brain-migrated domperidone inhibited D2R binding of [123I]IBZM. SPECT imaging is suitable for research to indirectly explore nose-to-brain drug delivery and locus-specific biological distribution.

Congenital sideroblastic anemia model due to ALAS2 mutation is susceptible to ferroptosis.

X-linked sideroblastic anemia (XLSA), the most common form of congenital sideroblastic anemia, is caused by a germline mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. In XLSA, defective heme biosynthesis leads to ring sideroblast formation because of excess mitochondrial iron accumulation. In this study, we introduced ALAS2 missense mutations on human umbilical cord blood-derived erythroblasts; hereafter, we refer to them as XLSA clones. XLSA clones that differentiated into mature erythroblasts showed an increased frequency of ring sideroblast formation with impaired hemoglobin biosynthesis. The expression profiling revealed significant enrichment of genes involved in ferroptosis, which is a form of regulated cell death induced by iron accumulation and lipid peroxidation. Notably, treatment with erastin, a ferroptosis inducer, caused a higher proportion of cell death in XLSA clones. XLSA clones exhibited significantly higher levels of intracellular lipid peroxides and enhanced expression of BACH1, a regulator of iron metabolism and potential accelerator of ferroptosis. In XLSA clones, BACH1 repressed genes involved in iron metabolism and glutathione synthesis. Collectively, defective heme biosynthesis in XLSA clones could confer enhanced BACH1 expression, leading to increased susceptibility to ferroptosis. The results of our study provide important information for the development of novel therapeutic targets for XLSA.

Uptake of nicotinic acetylcholine receptor imaging agent is reduced in the pro-inflammatory macrophage.

INTRODUCTION: Macrophages play a vital role in the development of atherosclerotic cardiovascular disease. Macrophages are functionally and phenotypically heterogeneous immune cells and commonly exist in two distinct or polarized subsets: pro-inflammatory M1 and anti-inflammatory M2 phenotypes. Previous reports suggest that stimulation of α7 or α4ß2 nicotinic acetylcholine receptors (nAChRs) in macrophages leads to an anti-inflammatory response. However, the biological link between nAChR expression on macrophages and the polarization state is unknown. Therefore, we evaluated the relationship between nAChRs and polarized macrophages in peritoneal macrophages and atherosclerotic plaques of apolipoprotein E knockout (ApoE-/-) mice. METHODS: Peritoneal macrophages isolated from mice were polarized into M1 and M2 macrophages, and the uptake of the nAChR-imaging agents, (R)-2-[11C]methylamino-benzoic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester ([11C]MeQAA) or 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy) pyridine ([18F]2FA), and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) was assessed. We also evaluated the accumulation of imaging agents in atherosclerotic plaques of ApoE-/- mice by autoradiography. After an autoradiogram was obtained, the same aortic tissue was used for immunohistochemical staining of CD68, inducible nitric oxide synthase (iNOS), and arginine-1. RESULTS: In an in vitro assay, the uptake of [11C]MeQAA or [18F]2FA was lower in M1 than in M0 and M2 macrophages. In comparison, the uptake of [18F]FDG was higher in M1 macrophages. Ex vivo autoradiography showed that [11C]MeQAA was localized to the extensive plaque area. By contrast, the accumulation of [18F]2FA and [18F]FDG was heterogeneous and found only in some plaques. Moreover, the expression of CD68 and iNOS was higher in [18F]2FA non-uptake than [18F]2FA uptake plaques. CONCLUSION: Macrophage polarization was related to nAChR expression, and α4ß2 nAChR expression was suppressed in the M1 macrophage. These findings suggest that nAChR imaging has the potential to identify the inflammatory status of atherosclerotic plaque.

Estrus Detection Using Background Image Subtraction Technique in Tie-Stalled Cows.

In this study, we determined the applicability of the background image subtraction technique to detect estrus in tie-stalled cows. To investigate the impact of the camera shooting direction, webcams were set up to capture the front, top, and rear views of a cow simultaneously. Video recording was performed for a total of ten estrous cycles in six cows. Standing estrus was confirmed by testing at 6 h intervals. From the end of estrus, transrectal ultrasonography was performed every 2 h to confirm ovulation time. Foreground objects (moving objects) were extracted in the videos using the background subtraction technique, and the pixels were counted at each frame of five frames-per-second sequences. After calculating the hourly averaged pixel counts, the change in values was expressed as the pixel ratio (total value during the last 24 h/total value during the last 24 to 48 h). The mean pixel ratio gradually increased at approximately 48 h before ovulation, and the highest value was observed at estrus, regardless of the camera shooting direction. When using front-view videos with an appropriate threshold, estrus was detected with 90% sensitivity and 50% precision. The present method in particular has the potential to be a non-contact estrus detection method for tie-stalled cows.

Differences in in vitro microglial accumulation of the energy metabolism tracers [18F]FDG and [18F]BCPP-EF during LPS- and IL4 stimulation.

The positron emission tomography probes 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 2-tert-butyl-4-chloro-5-{6-[2-(2-[18F]fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ([18F]BCPP-EF) are designed to evaluate glycolysis and oxidative phosphorylation, respectively, and are both used to estimate neuronal activity. However, previous studies have shown a discrepancy in these probes' accumulation in the compromised region, possibly due to the presence of activated microglia acting like deleterious or neuroprotective phenotypes. Hence, we evaluated lipopolysaccharide (LPS)- and interleukin 4 (IL4)-stimulated microglial uptake of [14C]2DG and [18F]BCPP-EF to give a new insight into the hypothesis that different uptake of [18F]FDG and [18F]BCPP-EF can be ascribed to the different metabolic pathways activated during microglial activation. LPS or IL4 stimulation increased the proinflammatory or anti-inflammatory marker gene expression in microglial cells. In LPS-stimulated cells, [14C]2DG uptake and glycolysis related gene expression were elevated, and [18F]BCPP-EF uptake was reduced. In IL4-stimulated cells, [18F]BCPP-EF uptake was increased, and [14C]2DG uptake was decreased. The expression of genes involved in glycolysis and mitochondrial complex I subunits was not changed by IL4 stimulation. The uptake of [14C]2DG and [18F]BCPP-EF differs in LPS- and IL4-stimulated polarized microglial cells. The present results suggest that the in vivo accumulation of metabolic tracers [18F]FDG and [18F]BCPP-EF can be influenced by the different aspects of neuroinflammation.

Conscious rat PET imaging with soft immobilization for quantitation of brain functions: comprehensive assessment of anesthesia effects on cerebral blood flow and metabolism.

BACKGROUND: Animal brain functions evaluated by in vivo imaging under anesthesia can be affected by anesthetic agents, resulting in incorrect assessment of physiological brain function. We therefore performed dynamic positron emission tomography (PET) imaging of conscious rats using recently reported soft immobilization to validate the efficacy of the immobilization for brain function assessments. We also determined the effects of six anesthetic agents-a mixed anesthetic agent (MMB), ketamine + xylazine (KX), chloral hydrate (Chloral), pentobarbital (PTB), propofol (PF), and isoflurane (IFL)-on brain function by comparison with conscious rats. RESULTS: The immobilization enabled 45-min dynamic [18F]FDG-PET acquisition with arterial blood sampling using conscious rats without the use of special techniques or invasive surgery. The spatial resolution and quantitativity of [18F]FDG-PET were not significantly lower for conscious rats than for anesthetized rats. While MMB, Chloral, PTB, and PF showed ubiquitous reduction in the cerebral metabolic rates of glucose (CMRglu) in brain regions, KX and IFL showed higher reductions in cerebellum and interbrain, and cerebellum, respectively. Cerebral blood flow (CBF) was reduced by MMB, KX, PTB, and PF; increased by IFL; and unaltered by Chloral. The magnitude of decrease in CMRglu and CBF for MMB were not larger than for other five anesthetic agents, although blood glucose levels and body temperature can be easily affected by MMB. CONCLUSION: The six anesthetic agents induced various effects on CMRglu and CBF. The immobilization technique presented here is a promising tool for noninvasive brain functional imaging using conscious rats to avoid the effects of anesthetic agents.

Multidisciplinary Team Care Delays the Initiation of Renal Replacement Therapy in Diabetes: A Five-year Prospective, Single-center Study.

Objective Although recent reports have highlighted the benefits of multidisciplinary team care (MTC) for chronic kidney disease (CKD) in slowing the progress of renal insufficiency, its long-term effects have not been evaluated for patients with diabetes mellitus (DM). We compared the renal survival rate between MTC and conservative care (CC). Methods In this five-year, single-center, prospective, observational study, we examined 24 patients (mean age 65.5±12.1 years old, men/women 18/6) with DM-induced CKD stage ≥3 in an MTC clinic. The control group included 24 random patients with DM (mean age 61.0±12.8 years old, men/women 22/2) who received CC. MTC was provided by a nephrologist and medical staff, and CC was provided by a nephrologist. Results In total, 10 MTC and 20 CC patients experienced renal events [creatinine doubling, initiation of renal replacement therapy (RRT), or death due to end-stage CKD]. During the five-year observation period, there were significantly fewer renal events in the MTC group than in the CC group according to the cumulative incidence method (p=0.006). Compared to CC, MTC significantly reduced the need for urgent initiation of hemodialysis (relative risk reduction 0.79, 95% confidence interval [CI] 0.107-0.964). On a multivariate analysis, MTC (hazard ratio [HR], 0.434, 95% CI 0.200-0.939) and the slope of the estimated glomerular filtration rate during the first year (HR, 0.429 per 1 mL/min/m2/year, 95% CI 0.279-0.661) were negatively associated with renal events. Conclusion MTC for DM-induced CKD is an effective strategy for delaying RRT. Long-term MTC can demonstrate reno-protective effects.

An attempt at estrus detection in cattle by continuous measurements of ventral tail base surface temperature with supervised machine learning.

We aimed to determine the effectiveness of estrus detection based on continuous measurements of the ventral tail base surface temperature (ST) with supervised machine learning in cattle. ST data were obtained through 51 estrus cycles on 11 female cattle (six Holsteins and five Japanese Blacks) using the tail-attached sensor. Three estrus detection models were constructed with the training data (n = 17) using machine learning techniques (random forest, artificial neural network, and support vector machine) based on 13 features extracted from sensing data (indicative of estrus-associated ST changes). Estrus detection abilities of the three models on test data (n = 34) were not statistically different among models in terms of sensitivity and precision (range 50.0% to 58.8% and 60.6% to 73.1%, respectively). The relatively poor performance of the models might indicate the difficulty of separating estrus-associated ST changes from estrus-independent fluctuations in ST.

Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane.

The high and persistent renal radioactivity levels after injection of radiolabeled low-molecular-weight polypeptides constitute a significant problem for their diagnostic and therapeutic applications, especially when they are labeled with metallic radionuclides. To improve the renal radioactivity levels of technetium-99m (99mTc)-labeled Fab fragments, a mercaptoacetyltriglycine (MAG3)-based new bifunctional chelating agent with a cleavable glycyl-phenylalanyl-lysine (GFK) linkage, MAG3-GFK-suc-TFP, was designed, synthesized, and evaluated. 99mTc-labeled Fab was obtained by reacting MAG3-GFK-Fab conjugate with 99mTc-glucarate. The GFK linkage remained stable in plasma but was cleaved by enzymes on the renal brush border membrane. The comparative biodistribution studies with indium-111 (111In)-labeled Fab using SCN-CHX-A″-DTPA showed that while both radiolabeled Fabs exhibited similar elimination rates from the blood, [99mTc]Tc-MAG3-GFK-Fab registered much lower renal radioactivity levels from 30 min post-injection onward due to the release and subsequent urinary excretion of [99mTc]Tc-MAG3-Gly. However, [99mTc]Tc-MAG3-GFK-Fab showed an increase in the intestinal radioactivity levels with the time that was not observed with 111In-labeled Fab. The analysis of the intestinal contents suggested the redistribution of [99mTc]Tc-MAG3-Gly to the intestine. The retrospective comparison of [99mTc]Tc-MAG3-GFK-Fab with the radiolabeled Fabs so far prepared under the identical concept suggested that some portion of [99mTc]Tc-MAG3-Gly was generated after the coated vesicle formation and they were excreted into the blood, and subsequently redistributed in the intestine via hepatobiliary excretion. In conclusion, MAG3-GFK-suc-TFP provided 99mTc-labeled Fabs that exhibit low renal radioactivity shortly after injection by the post-labeling procedure. The present study indicated that, contrary to our earlier proposal, the generation of the radiometabolites would proceed not only during the internalization process of the parental antibody fragments but also after coated vesicle formation. This study also showed that the intracellular behaviors of radiometabolites played crucial roles in the elimination rates and the routes of the radioactivity from the kidney.

Development of a High-Throughput Screening-Compatible Assay for Discovery of GPR3 Inverse Agonists Using a cAMP Biosensor.

While G-protein-coupled receptors (GPCRs) represent the largest class of cell surface proteins, there are ≥100 orphan GPCRs whose endogenous ligands are unknown. Accordingly, these could prove to be potential therapeutic targets for the pharmaceutical intervention of various diseases. Constitutively active orphan GPCRs are activated without ligands; thus, inverse agonists may be very useful pharmacological tools for inhibiting constitutive activity. However, in general, inverse agonist screening is considered more difficult to perform with high quality than antagonist screening, particularly due to the narrow assay window. We developed a high-throughput screening (HTS)-compatible assay to identify inverse agonists of GPR3. GPR3 is expressed in the central nervous system (CNS) and is known to be related to Alzheimer's disease and other CNS diseases. The GPR3 inducible cell line was established using T-REx 293 cells that stably expressed the tetracycline repressor protein, and the cAMP biosensor, GloSensor, was stably co-expressed. After optimization of the induction level of GPR3 and assay conditions, the GloSensor assay showed an approximately 20-fold signal-to-background ratio and high sensitivity. Using the HTS method, we successfully screened a library of hundreds of thousands of compounds for the inhibition of constitutive activity with good quality and excellent reproducibility. Finally, 35 compounds were identified as GPR3 selective inverse agonists. This inverse agonist screening approach using GloSensor in combination with the inducible expression of orphan GPCR indicates universal applicability to the search for inverse agonists of constitutively active orphan GPCRs.

Resolvin D1 suppresses inflammation-induced hyperexcitability of nociceptive trigeminal neurons associated with mechanical hyperalgesia.

7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (resolvin D1 [RvD1]) is biosynthesized from docosahexaenoic acid (DHA), and belongs to a novel family of lipid mediators showing remarkable anti-inflammatory effects; however, the effect of RvD1 on inflammation-induced hyperexcitability of nociceptive neurons under in vivo conditions remains to be determined. The present study, therefore, investigated whether under in vivo conditions, systemic administration of RvD1 could attenuate the inflammation-induced hyperexcitability of spinal trigeminal nucleus caudalis (SpVc) wide-dynamic range (WDR) neurons associated with hyperalgesia in rats. The threshold of escape from mechanical stimulation applied to the orofacial area in rats with complete Freund's adjuvant-induced inflammation was significantly lower than in naïve rats. The lowered mechanical threshold in rats with inflammation was returned to control levels following administration of RvD1 (3 ng/kg, i.p.) for 3 days. The mean discharge frequency of SpVc WDR neurons in rats with inflammation was significantly decreased after RvD1 administration for both non-noxious and noxious mechanical stimuli. Increased spontaneous discharge of SpVc WDR neurons in rats with inflammation was also significantly decreased after RvD1 administration. Noxious pinch-evoked afterdischarge frequency and occurrence in rats with inflammation was significantly diminished after RvD1 administration. Expansion of the receptive field in rats with inflammation also returned to control levels after RvD1 administration. These results suggest that administration of RvD1 attenuates inflammation-induced hyperexcitability of SpVc WDR neurons associated with inflammatory hyperalgesia. These findings support the idea that RvD1, derived from DHA, as well as DHA itself, are potential complementary or alternative therapeutic agents for the alleviation of inflammatory hyperalgesia.