Inducible nitric oxide synthase (iNOS) plays a crucial role in neuroinflammation, especially microglial activity, and may potentially represent a useful biomarker of neuroinflammation. In this study, we carefully defined a strategic plan to develop iNOS-targeted molecular PET imaging using (4-fluorophenyl)methanone  and control mice. Standard uptake value (SUV), total volume of distribution (Vt), and area under the curve (AUC) based on the [18F]FBAT PET signals were determined. The expression of iNOS was confirmed by immunohistochemistry (IHC) of brain tissues.  An 18F]FBAT was 2.2–3.1% (EOS), radiochemical purity was >99%, and molar radioactivity was 125–137 GBq/μmol. In vitro, [18F]FBAT rapidly and progressively accumulated in murine microglial BV2 cells exposed to LPS; however, [18F]FBAT accumulation was inhibited by aminoguanidine, a selective iNOS inhibitor. In vivo biodistribution studies of [18F]FBAT showed a significant increase in the liver and kidney on LPS-treated mice. At 3 h postinjection of LPS, in vivo, the [18F]FBAT accumulation ratios at 30 min post intravenous (i.v.) radiotracer injection for the whole brain, cortex, cerebellum, and brainstem were 2.16 ± 0.18, 1.53 ± 0.25, 1.41 ± 0.21, and 1.90 ± 0.12, respectively, compared to those of mice not injected with LPS. The mean area under the curve (AUC0-30min), total volume of distribution , and Ki (influx rate) of [18F]FBAT were 1.9 ± 0.21- and 1.4 ± 0.22-fold higher in the 3 h LPS group, respectively, than in the control group. In the pharmacokinetic two-compartment model, the whole brain Ki of [18F]FBAT was significantly higher in mice injected with LPS compared to the control group. Aminoguanidine, selective iNOS inhibitor, pretreatment significantly reduced the AUC0-30min and Vt values in LPS-induced mice. Quantitative analysis of immunohistochemically stained brain sections confirmed iNOS was preferentially upregulated in the cerebellum and cortex of mice injected with LPS.  At the end of synthesis, the yield of [18F]FBAT, and the preliminary in vitro and in vivo results demonstrated the feasibility of detecting iNOS activity/expression in LPS-treated neuroinflammation by noninvasive imaging with [18F]FBAT PET/MRI. An automated robotic method was established for radiosynthesis of [ Nitric oxide (NO) is a critical, unique mediator of a variety of physiological and pathological processes , 2. Two Nitric oxide (NO), the end product of metabolism catalyzed by the iNOS, is an important mediator of a variety of inflammatory diseases. When induced, iNOS generates significant amounts of NO (in the micromolar range), until the enzyme is degraded, sometimes hours later . Productα, IL-6, IL-8, and IL-1β—from host cells, especially mononuclear phagocytes [LPS is frequently employed to induce neuroinflammation in animal models. LPS induces the release of immunologically active mediators implicated in inflammatory diseases—including TNF-agocytes . Moreove11C]methyl ester ([11C]L-NAME) (iNOS IC50 = 20 μM) [11C]methylisothiourea , and S-(2-[18F]fluoroethyl)isothiourea  [50 = 0.16 μM) and selectivity for iNOS  [18F]fluorophenyl)-3,4-dihydro-1-isoquinolinamine ([18F]FFDI), which is based on FFDI, has been reported to act as a PET radiotracer for iNOS [18F]6-(2-fluoropropyl)-4-methyl-pyridin-2-amine ([18F]iNOS-9) was used as a PET tracer to image the activation of iNOS in a mouse model of lung inflammation fluorophenyl)methanone ([18F]FBAT) as a tracer to detect iNOS expression in mice injected with lipopolysaccharide (LPS) to induce brain inflammation. We assessed the potential of [18F]FBAT as a prognostic marker for the progression of neuroinflammatory disorders. [18F]FBAT was radiosynthesized from aryl pinacol boronates via the copper-mediated aromatic nucleophilic radiofluorination at an acceptable radiochemical yield and molar activity. Moreover, the [18F]FBAT PET findings were correlated with the results of immunohistochemistry. We anticipate that this study will not only advance our understanding of neuroinflammation diseases from the perspective of molecular imaging but also potentially help to develop novel tools to diagnose and guide the management of these diseases during the early progressive stages in humans.Because FFDI demonstrated similar or even superior potency in the cells to that of the standard iNOS inhibitors such as L-NMMA, FEITU, or MITU , therefo19F]FBAT reference standard were determined by NMR spectrophotometer . MS of [19F]FBAT reference standard were determined by LC/MS . NMR spectra of [18F]FBAT precursor were determined by NMR spectrophotometer . ESI-Mass of [18F]FBAT precursor were determined by LC/MS . ESI-Mass of [18F]FBAT precursor were determined by LC/MS . Quality control of the final product was conducted by analytical HPLC using a Varian 230 pump, Varian 325 UV/VIS (254 nm) detector (Varian Corp.), and Packard Radiomatic 150TR (BT cell) radiodetector . The amount of organic solvent was determined by gas chromatography (Varian Corp.).A Scanditronix MC 17 cyclotron was used for radionuclide production. The radiosynthesis was performed using a robotic system . Radiochemical yields were determined with a dose calibrator . The radioactive mixtures were purified via semipreparative high-performance liquid chromatography (HPLC) using a Varian 230 pump, Varian 325 UV/VIS detector , a pin-diode radiodetector , and a Rotavapor . NMR spectra of FBAT are presented in Schemes The synthetic routes of the precursor and authentic FBAT for 8FFBAT ar2O  dissolved in tetrahydrofuran (10 mL) was added to a solution of 1,4-dioxa-8-azaspiro[4.5]decane  and dichloromethane  under argon gas in an ice-bath; then after 10 min, a solution of 4-iodobenzoyl chloride  in dichloromethane (10 mL) was added, and the reaction mixture was stirred for 30 min at room temperature. Saturated NaHCO3 solution was added, and the organic layer was separated and dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica-gel column chromatography (60% ethyl acetate/n-hexane) to yield 5 g (96%) of compound 1 as a white solid. 1H NMR  δ = 7.75 , 7.13 , 3.98 , 3.82 , 3.46 , 1.78 , 1.63 ; MS (ESI+) m/z (%) 374.1 [M+H]+, 399.2 [M+Na]+.Boccompound 1  in hydrochloric acid ) was heated for 2 h at 90°C, cooled to room temperature, adjusted pH 10~11 with sodium hydroxide solution (10 wt%), diluted with dichloromethane and the organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by silica-gel column chromatography (50% ethyl acetate/n-hexane) to yield 1.3 g (49%) of compound 2 as a white solid. 1H NMR  δ = 7.80 , 7.22 , 3.86 , 2.50 ; MS (ESI+) m/z (%) 330.1 [M+H]+, 352.0 [M+Na]+.A solution of compound 1  and 2-amino-3,6-difluorobenzamidine dihydrochloride  in ethanol (5 mL) was refluxed overnight and cooled to room temperature, and the solid precipitate was filtered and purified by silica-gel column chromatography (5~10% methanol/dichloromethane) to yield 450 mg (76%) of compound 3 as a yellow solid. 1H NMR  δ = 7.74 , 7.14 , 7.00 , 6.33 , 4.22 , 3.70-3.45 , 2.20-1.90 , 1.70-1.60 ; MS (ESI+) m/z (%) 483.1 [M+H]+.A solution of 2O  dissolved in tetrahydrofuran (10 mL) was added to a solution of compound 3  and DMAP  in dichloromethane  in an ice bath. The reaction mixture was stirred overnight at room temperature, the solvent was evaporated under reduced pressure, and the residual solution was purified by silica-gel column chromatography (40% ethyl acetate/n-hexane) to yield 432 mg (70%) of compound 4 as a foamy yellow solid. 1H NMR  δ = 7.76 , 7.16 , 6.99 , 6.30 , 4.39 , 4.16 , 3.70-3.52 , 3.50-3.36 , 2.38-2.26 , 2.18-2.08 , 1.80-1.56 , 1.40 ; MS (ESI-) m/z (%) 681.7 [M-H]+.Boccompound 4 , palladium acetate , copper iodide , triphenylphosphine , cesium carbonate , and bis(pinacol)diboron  in acetonitrile (5 mL) was stirred for 16 h at room temperature, filtered through a celite pad, and concentrated under reduced pressure. The residue was diluted with dichloromethane and water; the organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by silica-gel column chromatography (50% ethyl acetate/n-hexane) to yield 176 mg (49%) of boron N-2-protected precursor as a light-yellow solid. 1H NMR  δ = 7.85 , 7.41 , 6.99 , 6.29 , 4.40 , 4.16 , 3.68-3.40 , 2.38-2.26 , 2.16-2.06 , 1.80-1.60 , 1.42 , 1.38 , 1.35 ; MS (ESI-) m/z (%) 681.4 [M-H]+  was added, the mixture was stirred, 2-amino-3,6-difluorobenzonitrile  was added gradually, and then, the solution was allowed to equilibrate to room temperature and refluxed for 18 h.The solution of 20 mL of methanol and metal sodium  was placed into a 250 mL three-port bottle under Ncompound 5 as a white solid product (1.831 g), yield 61.8%. 1H-NMR (CDCl3) 6.96-6.88 , 6.82 , 6.38-6.30 , 5.15 . LC-MS: calculated for C7H7F2N3O, 187.15; found [M+H] 188.1.The mixture was concentrated to oil that partitioned between ethyl acetate and 10% sodium hydroxide solution. The basic phase was separated and extracted three times with ethyl acetate, and the organic solution was washed three times with saturated brine and dried over sodium sulfate. The solvent was evaporated, and the product was purified by flash column chromatography on silica gel and eluted in DCM/MeOH = 100/1‐50/1 to obtain Compound 5  and wet Raney nickel (ca. 1.002 g) in ethanol (80 mL) were placed in a pressure bottle and stirred under 0.3 MPa hydrogen at 60°C for 8 h. The catalyst was removed by filtration and the solvent evaporated to give the product as oil, which was dissolved in ethanol (5 mL). HCl (1 N) in ether was added under stirring, and solid compound 5 was collected by filtration to give an off-white powder (2.028 g), yield almost 100%. 1H-NMR (CDCl3) 9.48 , 9.24 , 7.26-7.18 , 6.48-6.41 . LC-MS: calculated for C7H7F2N3, 171.15; found [M+H] 172.0.p-toluenesulfonic acid  was heated and refluxed in 350 mL of toluene for 6 h; water from the reaction was removed by azeotropic distillation of the water separator. After cooling to room temperature, 2 M aqueous sodium hydrate was added to adjust the pH to 14, the organic layer was separated, the aqueous layer was extracted with toluene, and the toluene layer was combined, followed by drying on anhydrous sodium carbonate. After removing sodium carbonate by filtration, the solvent was distilled off under reduced pressure, and the residue was distilled under reduced pressure to obtain a colorless liquid . 1H-NMR (CDCl3) 3.96 , 2.94-2.90 , 1.68-1.64 , 1.42 . LC-MS: calculated for C7H13NO2, 143.19; found [M+H] 144.1.A mixture of 4-piperidone monohydrate hydrochloride , ethylene glycol , and Compound 7 , triethylamine , DMAP , and methylene chloride (20 mL) were added to a three-mouth bottle in an ice-salt bath under N2 atmosphere. A methylene chloride solution of 4-fluorobenzoyl chloride  was added dropwise while keeping the temperature below 0°C; then, the solution was equilibrated to room temperature and stirred overnight. The DCM was diluted and washed with brine, and the organic extract was dried over magnesium sulfate and filtered. The solvent was evaporated under reduced pressure, and the remaining mixture was separated, dried, and concentrated. The crude product was purified by flash column chromatography on silica gel and eluted with DCM/MeOH = 100/1 to obtain compound 8 as an off-white solid (1.610 g). The product was recrystallized in ethyl acetate and n-hexane to obtain 1.152 g of white crystals . 1H-NMR (CDCl3) 7.41-7.39 , 7.12-7.09 , 3.99 , 3.82 , 3.51 , 1.72-1.69 . LC-MS: calculated for C14H16FNO3, 265.28; found [M+H] 266.0.Compound 6 hydrochloride , compound 8 , and 20 mL isopropyl alcohol were added to a 50 mL single-neck bottle under N2 atmosphere. The reaction mixture was refluxed overnight and cooled to precipitate a large amount of yellow solid. The mixture was filtered, and the filter cake was washed with ether to obtain 0.382 g of yellow solid. The solid was dissolved in 10 mL of methanol and adjusted to pH 8 with ammonia, and the mixture was extracted twice with dichloromethane. The organic phase was washed with saturated sodium chloride solution, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel, eluted with DCM/MeOH (20/1 v/v) to obtain the product, which was recrystallized in dichloromethane and n-hexane to obtain the product (0.310 g) as an off-white solid . 1H-NMR (DMSO) 8.31-8.28 , 7.66-7.63 , 7.18-7.10 , 6.39-6.31 , 4.26-4.22 , 3.45-3.39 , 3.32-3.26 , 1.87-1.68 . LC-MS: calculated for C19H17F3N4O, 374.37; found [M+H] 375.2  at 120°C and coevaporated to dryness with CH3CN (7 × 0.5 mL). Cu(II)(OTf)2 (7.3 mg) and 0.1 mL pyridine/0.6 mL dry DMF were added to the dried [18F]KF, and dry air was delivered to the vial for 1 min.No-carrier-added (NCA) aqueous [18F]FBAT (2.0 mg in 0.5 mL of DMF) was added to the dried [18F]KF, and dry air was delivered to the vial for 1 min. The labeling reaction was carried out in a heating block station at 110°C for 10 min. The reaction mixture was passed through a silica plus Sep-Pak cartridge that had been preconditioned with 5 mL of CH2Cl2 to keep the cartridge wet. Then, the vial was rinsed with 1.5 mL of CH3OH/CH2Cl2 (2/3 v/v), the solution was passed through a silica plus Sep-Pak cartridge, another 1.5 mL of CH3OH/CH2Cl2 was passed through the silica plus Sep-Pak cartridge, and the eluate was reduced to 0.4 mL in a heating block station at 65°C under a stream of nitrogen (200 mL/min) in a vacuum. One milliliter of 6 N HCl was added, the solution was heated at 110°C for 10 min and cooled to room temperature, and 1 mL of 6 N NaOH was added to neutralize the solution. The resulting solution was passed through a 0.22 μm PVDF membrane filter (Millipore) into a 5 mL Reacti-vial. The reaction scheme is shown in The precursor of [18F]FBAT product, a filter check station that checked the integrity of the 0.22 μm membrane filters, a solvent rack station, and a water cooling station.All procedures described above were performed using a robotic system . A diagram of the synthetic system is shown in 18F]FBAT was purified via semipreparative HPLC on a C18 guard column  followed by a RP-C18 semipreparative column  and eluted with acetonitrile/(0.1% triethylamine/H2O)  pH 9.0 at a flow rate of 10 mL/min. The [18F]FBAT fraction was transferred to a round-bottomed flask and evaporated to dryness using a rota-evaporator. The formulation was prepared by adding 2 mL of physiological saline (0.9%) to reconstitute the final [18F]FBAT product. The saline solution of [18F]FBAT was passed through a 0.22 μm filter  into a sterile, pyrogen-free vial.Crude [18F]FBAT were determined via analytical HPLC on a reverse phase column  and eluted with acetonitrile/(0.025% triethylamine/H2O)  at a flow rate of 1 mL/min and detected using a UV-VIS detector (254 nm) and radio detector. The amount of organic solvent was determined by gas chromatography.The radiochemical purity and molar activity of FBAT at 0.37 MBq/mL for 15, 30, or 60 min, harvested by gentle scraping, and pelleted by centrifugation at 1500 g for 2 min. The weight of the cell pellet and 0.1 mL of the radioactive supernatant was determined. A Packard 5500 gamma counter  was used to quantify radioactivity as cpm/g cells or cpm/mL media, respectively. Cell-to-medium radioactivity concentration ratios were calculated and plotted over time to evaluate the kinetics of accumulation of the radiotracer.BV2 cells were cultured as described above, washed three times with fresh medium, pretreated with a selective iNOS inhibitor, aminoguanidine (AMG),  for 1 h, and then treated with cell culture media containing LPS and the inhibitor. At the indicated time points, the cells were washed thrice with fresh cell culture media, then incubated in fresh cell culture media continuing the radiotracer [18F]FBAT in BV2 cells before or after iNOS inhibitor versus NO production was also assessed.iNOS is the enzyme responsible for NO generation in BV2 cells , 21; thead libitum and were housed under controlled room temperature (22 ± 2°C) and humidity (55-65%) under a 12 : 12 h light-dark cycle. The dark cycle lasted from 19:00 to 7:00.All animal handling procedures were approved by Yang-Ming University Institutional Animal Care and Use Committee (IACUC No. 1050910), and animal study was performed according to the Guidelines for Animal Experimentation of National Yang-Ming University. For metabolism study, the ten-week-old male Sprague-Dawley rat (255 ± 12.3 g) or eight-week-old male C57BL/6 mice (22 ± 0.4 g) received food and water n = 18, weight = 25 ± 0.6 g) were intratracheally administered a bolus containing 5, 10, or 15 mg/kg LPS . Behavior, breathing rate, appetite, and mortality rate were recorded over 48 h. The group receiving 10 and 15 mg/kg LPS exhibited mild to moderate clinical and physical symptoms of adult respiratory distress syndrome (ARDS) within 24 h of administration of LPS. All animals exhibited chills; the mortality rates for the 5, 10, and 15 mg/kg LPS groups were 16.6%, 50%, and 83%, respectively. The mortality rates reduced to 0%, 40%, and 50%, respectively, when the animals were kept on heating pads until they recovered; these animals were less mobile and had decreased appetite. Thus, 5 mg/kg was determined as a suitable dose of LPS to induce moderate neuroinflammation in mice.We performed a preliminary study to identify the dose of LPS that induces moderate neuroinflammation in mice. Male C57BL/6 mice from BioLASCO , and six mice were injected i.p. with aminoguanidine (Sigma-Aldrich) 30 min prior to 5 mg/kg LPS. In vivo imaging was performed 3 h or 24 h after injection of LPS; then, the mice were immediately humanely euthanized and the brains were removed for immunostaining.For the imaging experiments, twelve C57BL/6 mice were injected n = 3) were anesthetized with 2% isoflurane (in oxygen) and injected with 194.87 MBq ± 22.61 MBq of [18F]FBAT in the tail vein. Plasma was extracted from heparinized blood samples (0.25 mL) with 3x volumes of acetonitrile and analyzed with radio-HPLC; mobile phase: 40% MeCN/0.10% TEA in water at 1 mL/min. This method affords retention time of [18F]FBAT which was 18.1–18.9 min. Fractions of [18F]FBAT and metabolites were calculated for each sample based on the area under each peak. The radioactivity concentration in the whole blood and plasma was assayed using a gamma counter .Adult Sprague-Dawley rats (n = 30) were anesthetized with 2% isoflurane (in oxygen) and injected with [18F]FBAT  via the tail vein. Dynamic PET images were obtained in fully three-dimensional list mode for 30 min using a small animal SuperArgus 2r PET system  or 7T PETMR Inline . Images were acquired every 10 s for 12 images, 60 s for three images, 300 s for three images, or 600 s for four images. The anatomical structure of the brain was imaged using T2 MRI. The MRI sequences included 0.5 mm thick T2 Turbo RARE high-resolution images .Mice  administered a bolus of aminoguanidine (50 mg/kg i.p.) 30 min prior to administration of [18F]FBAT. The time course of [18F]FBAT radioactivity in the brain after administration of the iNOS inhibitor was assessed and compared to that of the mice in the LPS 3 h group.After determining the optimal imaging time point for [18F]FBAT was estimated from the mean pixel values within the region of interest (ROI) corresponding to MR images of various organs and regions of the brain. ROI for carotid artery, heart muscle, lung, liver, spleen, and kidney were defined and time activity curves (TACs) generated. TACs for whole brain, cortex, cerebellum, and brainstem were also determined as described above.Images were reconstructed by the Fourier rebinning algorithm and two-dimensional filtered back projection using a ramp filter with a cutoff at Nyquist. The regional radioactivity concentration (kBq/cc) of FBAT input function in uncorrected blood plasma. The corresponding TACs for [18F]FBAT were derived by applying images from mice administered [18F]FBAT to the image-derived plasma TAC. Model parameters were estimated for influx constant k1 (mL/cm3/min−1), efflux (k2) (min−1) rate of radioligand diffusion between plasma and brain compartment. Exchange between compartments k3 (min−1) and k4 (min−1) was also estimated. The net influx constant, Ki (min−1), parameter that describes the rate of binding to the iNOS was calculated asA time activity curve (TAC) for a carotid artery region of interest was used to determine the dynamic PET image-derived  range. Immunostaining intensity was manually measured in the region of interest (ROI). The median  percentage score for each group  was calculated as the sum of the individual number of slides within the group.t-tests with Welch's correction using GraphPad Prism 8 . P < 0.05 was considered significant.Data are presented as the mean ± SD values for each group, and were compared using one-way ANOVA and the post hoc Bonferroni test or unpaired 19F]FBAT is shown in Schemes The preparation of FBAT precursor and authentic product 9FFBAT is18F]FBAT preparation using a robotic system. We found that the optimization of precursor/Cu(OTf)2 ratio was 1.46 and temperature was 110°C . A silica plus Sep-Pak cartridge was used to trap the unreacted [18F]fluoride. The total synthesis time was 200 min. The retention time (tR) of [18F]FBAT in semipreparative HPLC was 10.07 min  of [18F]FBAT . Thereafter, the accumulation of [18F]FBAT reached a plateau in control BV2 cells (black line) around a cells-per-medium concentration ratio of 100. In contrast, in LPS-induced BV2 cells (red line), [18F]FBAT accumulation continued to increase up to 1 h and thereafter reached a plateau at a cells-per-medium concentration ratio of 180–200. [18F]FBAT accumulation at 60 min was more than 1.7-fold higher in LPS-induced BV2 cells than control BV2 cells (18F]FBAT accumulation (P < 0.05)  clearance kinetics after i.v. injection . Clearance of [18F]FBAT from their circulation exhibited a biexponential kinetics with half-lives of 1.05 ± 0.58 min and 24.9 ± 7.46 min, respectively , determined from the mean pixel activity within the ROI placed over the carotid region. The time activity curve of carotid as an input function was applied for Logan plot to estimate total volume distribution of [18F]FBAT or pharmacokinetic two-compartment model. One of the aims of the study was to preliminarily measure and analyze [18F]FBAT accumulation in the three major regions: cortex, cerebellum, and brainstem. Whole brain was used as an average uptake per voxel in all regions. PET imaging revealed rapidly cross BBB and accumulation of [18F]FBAT in the whole brain and, to a lesser degree, in the cortex, cerebellum, and brainstem. After LPS induction, the radioactivity of [18F]FBAT in the whole brain, cortex, cerebellum, and brainstem reflected the concentration of [18F]FBAT in the blood pool and was surprisingly high at 3 h postinjection of LPS. Radioactive accumulation of [18F]FBAT was 1.43 ± 0.11 SUV in the whole brain, 0.90 ± 0.07 SUV in the cortex, 0.60 ± 0.03 SUV in the cerebellum, and 0.98 ± 0.09 SUV in the brainstem  were 2.16 ± 0.18 (∗P < 0.05) in the whole brain, 1.53 ± 0.05 (∗P < 0.05) in the cortex, 1.41 ± 0.21 (∗P < 0.05) in the cerebellum, and 1.91 ± 0.12 (∗P < 0.05) in the brainstem. The quantitative measures of [18F]FBAT accumulation at 30 min post i.v. injection of [18F]FBAT at various time points in the LPS-induced model are summarized in The parametric images were used to determine the standardized uptake value ratios (SURs) after normalization to the control group . SURs  using the mean area under the curve of the tissue radioactivity from 0 to 30 min . Integrated activity (0-30 min) showed more [18F]FBAT retention in the LPS 3 h group in comparison to control or LPS 24 h mice  for [18F]FBAT in brain regions was 0.40 ± 0.02 , the net influx rate constant  was also calculated using two-tissue compartment model. The whole brain Ki of [18F]FBAT (0.007 ± 0.003 min−1) in the LPS group was significantly higher than that of controls (4.45E − 06 ± 2.01E − 06 min−1). The influx  and efflux  rate constants of [18F]FBAT in the whole brain were no difference between the control and LPS groups which are consistent with the moderate lipophilicity of the radiotracer. The [18F]FBAT k1, k2, and k3 rate constants and Ki for various brain structures are presented in Additional to using Logan graphical analysis to calculate volume distribution  30 min before LPS induction significantly reduced [18F]FBAT accumulation by decreasing iNOS expression, with corresponding SUVs of 0.31 ± 0.08, 0.32 ± 0.09, 0.31 ± 0.09, and 0.35 ± 0.10 in the whole brain, cortex, cerebellum, and brainstem, respectively . Immunostaining for iNOS , whereas iNOS immunoactivity in the cerebellum was not significantly different between the LPS 3 h and 24 h groups (#P < 0.05), but not in the cerebellum, compared to the LPS 3 h group.Quantitative analysis of the immunostained sections confirmed iNOS immunoactivity preferentially accumulated in viable portions of the cerebellum and cortex of mice with LPS-induced inflammatory responses  were very poor. It would be very difficult to carry out radiofluorination by substituting nitro group on the benzene ring if there were no electron-withdrawing groups in the nitrobenzene ring. Recent advances have expanded the scope of nucleophilic aromatic radiofluorination by using triarylsulfonium diarylselenone [18F]FBAT by FBAT.eactions , 24. Theeactions , though selenone  26] or  or 18F-lselenone –29. The selenone  which pr]FBAT by  a boron us study . However18F]F-NOS has been previously investigated and demonstrated that iNOS activity correlated significantly with lung tissue measurements in healthy volunteers and in myocardial tissue of orthotopic heart transplant patients undergoing surveillance for rejection [in vivo using [18F]F-6-(2-fluoropropyl)-4-methyl-pyridin-2-amine, both in a LPS-induced lung inflammation of mouse model and endotoxin-induced lung inflammation in healthy volunteers. Significantly higher [18F]F-6-(2-fluoropropyl)-4-methyl-pyridin-2-amine uptake was reported in the lungs of mice treated with LPS than control mice FBAT synthesis and the first histologically validated study of [18F]FBAT for in vitro and in vivo PET imaging. We demonstrated that LPS-induced microglial BV2 cells, which are the most frequently used in vitro model of primary microglia [18F]FBAT than control cells. Preliminary semiquantitative measurements (metabolite-uncorrected) of [18F]FBAT-PET demonstrated rapid, high accumulation of [18F]FBAT in the brains of mice with LPS-induced brain inflammation. We also observed significantly higher accumulation of [18F]FBAT in the brain at 3 h after LPS stimulation than at 24 h after LPS stimulation.To our knowledge, this is the first report of FBAT. However, most of the iNOS radioligands available are also known to show similar metabolism. For example, [11C]L-NAME revealed that at 10 min postinjection 65% of the radioactivity was [11C]methanol (metabolite) in dog [18F]9 [18F]FBAT use since it makes its quantification more difficult. Nevertheless, the contribution of the major metabolites of [18F]FBAT circulating in blood which led to accumulation of [18F]FBAT-derived radioactivity in the brain needs to be accounted for in future investigations.Our metabolism study showed a rapid decrease of the parent fraction in plasma where roughly 50% of the metabolites was detected at 15 min postinjection of 9 . This mein vivo biodistribution studies showed that the highest uptake in both groups was observed in the liver and kidneys, which are likely to be the major metabolic and/or excretory sites for [18F]FBAT. The rapid whole-body redistribution and fast renal clearance of [18F]FBAT from circulation make this radiotracer especially suitable for imaging of the chest/abdominal area, specifically of the lung. No significant accumulation of [18F]FBAT-derived radioactivity was detected in the skeletal structures up to 30 min postinjection of [18F]FBAT, which suggests that the radiotracer was not catabolized or defluorinated in vivo. Our results of whole-body biodistribution of [18F]FBAT-derived radioactivity were in agreement with iNOS distribution in organs in male BLAB/c mice that reported iNOS mRNA and protein expression 6 h after LPS stimulation was observed in many organs. Among them, the highest iNOS expression was in the lungs followed by that in the kidneys, heart, gut, and liver [The results of nd liver .11C]methylisothiourea ([11C]MITU) and S-(2-[18F]fluoroethyl)isothiourea ([18F]FEITU) in mature female Sprague-Dawley rats in control and pretreated with 10-20 mg/kg LPS, 6 h before tracer injection of radiotracers FBAT to quantify [18F]FBAT accumulation in the brain from the PET images. The AUC0-30min and Vt values of [18F]FBAT in the whole brain were averagely 1.9 ± 0.21- and 1.4 ± 0.22-fold lower in control mice than in the LPS 3 h group. After treatment with selective iNOS inhibitor, AUC0-30min and Vt values showed a statistically significant reduction in AUC and Vt of [18F]FBAT in the LPS group .In this initial evaluation, we used AUC and 18F]FBAT and dynamic contrast-enhanced MRI (DCE-MRI) assess dose dependence of LPS on BBB permeability/disruption in rodent model or neuroinflammation produced by intracerebral injection of LPS.Further studies such as coupling imaging of PET [18F]FBAT radioactivity. The PET/MR results indicated that the radioactivity of [18F]FBAT correlated with the levels of iNOS expression induced by cellular inflammatory responses in the brain observed using quantitative immunohistochemistry (QIHC). Pretreatment of animals with the selective iNOS inhibitor aminoguanidine significantly decreased [18F]FBAT accumulation in all structures of the brain, consistent with the QIHC studies.The higher resolution 7T PET/MR system was used to enable more detailed assessment of the heterogeneous accumulation of [in vivo PET imaging studies with [18F]FBAT in mouse brain expressing different levels of iNOS after LPS inducement are more selective. Predominant accumulation of [18F]FBAT in iNOS-expressing brain regions could be implied to be responsive to therapy with selective iNOS inhibitor  -ROStrace PET imaging targeted to superoxide and reported the level of neuroinflammation induced by LPS (5 mg/kg) was strongly associated with the severity of the pain and distress scores FBAT in the systemic circulation crosses the BBB and directly activates cells within the CNS. Based on the current results, we consider that [18F]FBAT crosses the BBB and cell membranes bidirectionally by nonfacilitated diffusion, due to its physicochemical characteristics (Log P 1.40). Therefore, the rate constants of [18F]FBAT influx (k1FBAT) and efflux (k2FBAT) across the BBB and cell membranes are assumed to be high and not rate limiting. The critical issue of whether LPS actually disrupts the BBB and results in overestimation of [18F]FBAT accumulation in the brain can be answered using the pharmacokinetic two-compartment model. Based on our preliminary evaluation, [18F]FBAT influx (k1FBAT) and efflux (k2FBAT) were similar with or without LPS induction; however, k3 was significantly different between LPS-injected and control mice. This suggests that the higher [18F]FBAT Vt observed in the brain of LPS-injected mice is most likely due to increased expression of iNOS in the brain parenchyma. Also, due to the low brain uptake of the tracer (1 to 2 SUV), cerebral passage of [18F]FBAT does not depend on cerebral blood flow. Thus, Vt represents the most robust parameter for quantifying [18F]FBAT uptake and is not affected by changes in cerebral blood flow. Nevertheless, further metabolite analysis of [18F]FBAT in the brain and knowledge of the dose-response relationship between LPS and [18F]FBAT accumulation in the brain are required to validate the implications of BBB disruption by LPS.Another critical issue that needs to be addressed is whether [18F]FBAT from a boron ester precursor in a hot cell. A HPLC method was employed to purify the product. The radiochemical purity of [18F]FBAT was greater than 99%, and the corrected radiochemical yield was about 2.2-3.1% (EOS). Preliminary in vitro and in vivo evaluations demonstrated [18F]FBAT could potentially be used to detect iNOS activity and expression in LPS-induced neuroinflammation. Further investigations, including optimization of the radiochemical yield and detailed brain region-specific distribution in animal PET/MRI imaging research applications, are currently underway in our laboratory.An automated robotic method was established for radiosynthesis of [


Recently, the steroidal 2-methoxyestradiol was shown to inhibit nuclear translocation of the nuclear factor of activated T cells (NFAT). We therefore investigated 2-methoxyestradiol for inhibition of Ca2+ entry in T cells, screened a library of 2-methoxyestradiol analogues, and characterized the derivative 2-ethyl-3-sulfamoyloxy-17β-cyanomethylestra-1,3,5(10)-triene (STX564) as a novel, potent and specific SOCE inhibitor. STX564 inhibits Ca2+ entry via SOCE without affecting other ion channels and pumps involved in Ca2+ signaling in T cells. Downstream effects such as cytokine expression and cell proliferation were also inhibited by both 2-methoxyestradiol and STX564, which has potential as a new chemical biology tool.T cell activation starts with formation of second messengers that release Ca •2-Ethyl-3-sulfamoyloxy-17β-cyanomethylestra-1,3,5(10)-triene is a SOCE inhibitor.•novel inhibitor antagonizes SOCE and subsequent signaling events in different T cells•3 receptor, KV channels or SERCA pumpsno antagonist effects on IP Althoiewed in . Furtheriewed in . Therebyiewed in . Recentliewed in . In the 2+ entry in T cells, screened a library of 2ME2 analogues, and characterized STX564 as novel, potent and specific SOCE inhibitor, similar in efficacy to the known SOCE inhibitor Synta66 i) from the ratio data:Finally, every measurement was calibrated using 0.1% . The fluorescence intensities of deleted data points were interpolated from those just before and after the deleted time points.s 224 nM . R is thv/v) for all conditions.Estradiol, 2ME2 and 2ME2 derivatives were dissolved in DMSO at a concentration of 50 mM and stored at −20 °C for up to 6 months. For concentration-response curves, stock solution was freshly diluted with DMSO to obtain the same DMSO concentration of 0.2%  were calculated and subtracted from the basal Ca2+ concentration that represents the mean Ca2+ concentration before the first compound addition. These plateau Ca2+ concentrations were normalized to 100% (DMSO condition of every data set) and 0% . The compound concentrations were then log transformed. Finally, the data were fitted to a corresponding nonlinear fit of variable slope using Prism 6 .For calculating IC2.62+ quenching of Fura2 was used to analyze influx of divalent cations into Jurkat T cells. Like for Ca2+ measurements, 106 loaded Jurkat T-lymphocytes were pelleted and Ca2+ buffer was exchanged for nominal Ca2+ free buffer (see above). Instead of Ca2+, Mn2+ was added 1220 s after the start of the measurement. To investigate Mn2+ quenching independently of any effects of Ca2+, Fura2 was excited at its isosbestic point, which was experimentally determined to be 357 nm. The emission wavelength was set to 495 nm. In contrast to the Ca2+ measurement experiments, these measurements were not calibrated.Mn2+ addition, there was a very fast drop in Fura2 fluorescence in all experiments. This drop lasted for about 10 s and was probably due to quenching of extracellular Fura2. This phase was not considered in the calculation of the initial slope. The data after this rapid drop were fitted to a one phase exponential decay with the following equation:For quantitative analysis, the velocity of quenching was calculated as the initial slope. After Mny0 is the fluorescence at the start of the decay, P is the theoretical fluorescence at the end of the decay at infinite times, K is the rate constant, and x is the time.The first derivative of this equation was used to calculate the slope at 1230 s:2+ addition due to bleaching. The fluorescence before Mn2+ addition was fitted to a straight line using the following equation:In all experiments, fluorescence gradually decreased before Mnm is the slope, x is the time and c is the y-intercept. To not overestimate the initial slope, the slope of this linear fit was subtracted from the initial slope of the exponential decay. So the initial slope (si) after Mn2+ addition was calculated using the following equation:2.76 Jurkat T–lymphocytes were pelleted at 400 to 450 ×g for 5 min at room temperature and pellets were dissolved in 500 μl RPMI 1640 medium (Gibco) without phenol red containing l-glutamine, supplemented with 7.5% (v/v) NCS. 30 μg plasmid-DNA (G-GECO1.2-ORAI1) was transferred into a 4 mm electroporation cuvette  and the Jurkat T-lymphocytes were added. G-GECO1.2-ORAI1 was a gift from Michael Cahalan, USA (Addgene plasmid # 73562)  after IP3 addition was subtracted from the mean [Ca2+] between 25 and 75 s before IP3 addition (dPeak). For analyzing SERCA inhibition, the data after compound addition, namely thapsigargin, STX564, Synta66 or DMSO as a vehicle control, and the decay after ATP addition was fitted to a ‘plateau followed by one phase exponential decay’ using GraphPad Prism. The rate constant K of the fitted data was used as a quantitative read-out for SERCA pump activity and its inhibition.As a quantitative read-out for inhibition of IP2.9w/v) PLL solution  diluted 1:50 with high-purity water  for 30 min. Coated culture dishes were rinsed twice with high-purity water, dried at room temperature under sterile conditions and then stored at 4 °C for up to 1 week. Just before starting the experiments, coated culture dishes were pre-warmed for 5 min at room temperature. A recording chamber  that was preliminary coated with a thin layer of baysilone-paste  of medium viscosity was placed into the coated cell culture dish. About 105 Jurkat T-lymphocytes were placed into this recording chamber and were allowed to settle down for 5–10 min. Afterwards the cells were rinsed twice with 200 μl extracellular buffer (composition: see below next paragraph) and finally taken up in 200 μl extracellular buffer containing 200 nM thapsigargin as well as either K+ channel inhibitors, STX compounds or 0.2% (v/v) DMSO as a vehicle control. The cell culture dish was placed in a custom-made holder placed on an inverted microscope (Zeiss AxioVert 100). Microelectrodes were manually pulled from 1.5 mm diameter borosilicate glass capillaries  and firepolished via a Flaming Brown micropipette puller , resulting in pipette resistances between 2.7 and 5.1 MΩ (mean: 3.9 MΩ). The microelectrodes were filled with intracellular solution (composition: see below next paragraph). The holding potential was set to −60 mV. Extracellular buffer for KV measurement contained 128 mM CH3SO3Na, 4 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose; pH 7.4, NaOH .V channels contained 125 mM aspartic acid, 5 mM MgCl2, 2 mM MgSO4, 15 mM HEPES, 12 mM BAPTA  were compensated by Patchmaster . C-slow and series resistance was compensated before every sweep. The voltage was applied to the cells and the currents were recorded via an EPC9/10 amplifier at a sampling rate of 20 kHz.Intracellular buffer for measuring Kied from ). For me2.10+ T cells retrovirally transduced to express enhanced GFP (TMBP cells) were established as previously reported i is essential for downstream signaling events of T cell activation, inhibition of Ca2+ entry is likely the mechanism by which 2ME2 decreases NFAT translocation, T cell proliferation and cytokine production [2+]i upon exposure to STX564, we excluded experimentally (i) inhibition of IP3 evoked Ca2+ release, (ii) inhibition of SERCA activity, and (iii) inhibition of KV as discussed above [We clearly demonstrated that the hormone estradiol, its non-estrogenic metabolite 2ME2, and its synthetic analogue STX564 inhibit Caoduction  as well oduction . Among ted above .+/K+ ATPases in human and mouse cardiac myocytes [+ channels were described, e.g. activation [V channels nor SERCA pumps.While it was shown that estradiol activates (i) Namyocytes ,43 (ii) myocytes , and . Actionl target ,54, modul target ,54, whicl target . Finallyl target . Howevereceptors .2+ release channels, SERCA pumps nor KV channels. This compound should serve as an attractive new chemical biology tool for future studies on SOCE, but also in autoimmune disease models.In conclusion, we characterized the 2ME2 derivative STX564 as a novel potent and specific SOCE inhibitor affecting neither CaSource data are available from the corresponding author upon request.10.13039/501100001659Deutsche Forschungsgemeinschaft (DFG), Germany,  and by the Hertie Stiftung, Germany . BVLP is a 10.13039/100010269Wellcome Trust Senior Investigator .This work was supported by the Anke Löhndorf: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration. Leon Hosang: Methodology, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Wolfgang Dohle: Investigation, Resources, Writing – review & editing. Francesca Odoardi: Conceptualization, Methodology, Validation, Investigation, Writing – original draft, Writing – review & editing, Supervision, Project administration. Sissy-Alina Waschkowski: Formal analysis, Investigation, Data curation, Writing – review & editing. Anette Rosche: Formal analysis, Investigation, Data curation. Andreas Bauche: Formal analysis, Investigation, Data curation. Riekje Winzer: Investigation. Eva Tolosa: Investigation. Sabine Windhorst: Investigation. Stephen Marry: Methodology, Formal analysis, Investigation, Data curation, Writing – review & editing. Alexander Flügel: Conceptualization, Validation, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition. Barry V.L. Potter: Conceptualization, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition. Björn-Philipp Diercks: Methodology, Formal analysis, Investigation, Data curation, Writing – review & editing. Andreas H. Guse: Conceptualization, Validation, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition.The authors declare no competing interest.


Chronic kidney disease (CKD) is one of the main long-term prognosis factors in methylmalonic acidemia (MMA), a rare disease of propionate catabolism. Our objective was to precisely address the clinical and biological characteristics of long-term CKD in MMA adolescent and adult patients.In this retrospective study, we included MMA patients older than 13 years who had not received kidney and/or liver transplantation. We explored tubular functions, with special attention to proximal tubular function. We measured glomerular filtration rate (mGFR) by iohexol clearance and compared it to estimated glomerular filtration rate (eGFR) by Schwartz formula and CKD-EPI.2 (23.3 to 105 mL/min/1.73 m2). Ten out of 13 patients had mGFR below 90 mL/min/1.73 m2. No patient had significant glomerular proteinuria. No patient had complete Fanconi syndrome. Only one patient had biological signs suggestive of incomplete proximal tubulopathy. Four out of 13 patients had isolated potassium loss, related to a non-reabsorbable anion effect of urinary methylmalonate. Both Schwartz formula and CKD-EPI significantly overestimated GFR. Bias were respectively 16 ± 15 mL/min/1.73 m2 and 37 ± 22 mL/min/1.73 m2.Thirteen patients were included (M/F = 5/8). Median age was 24 years (13 to 32). Median mGFR was 57 mL/min/1.73 mCKD is a common complication of the MMA. Usual equations overestimate GFR. Therefore, mGFR should be performed to inform therapeutic decisions such as dialysis and/or transplantation. Mild evidence of proximal tubular dysfunction was found in only one patient, suggesting that other mechanisms are involved.The online version contains supplementary material available at 10.1186/s13023-021-01851-z. MUT gene, encoding MCM, or to mutations in MMAA (CblA), MMAB (CblB) and occasionally MMADHC (CblD) genes, involved in the metabolism of its cofactor adenosylcobalamin   than in mut0  and cblA patients   had plasma potassium at 3.7 mmol/L despite potassium replacement therapy (3.6 g/day) and CKD stage 3b (Table p = 0.004)  . Serum potassium was significantly lower in mut.7 mmol/LThe biological results are summarized in Table p = 0.054).None of the patients had past history of stones or gout. None of the patients had hypouricemia suggestive of proximal tubulopathy. As expected, serum uric acid tended to be inversely proportional to mGFR . Two patients (#07 and #08) had a slight decrease of ionized calcemia below 1.15 mmol/ L, related to a 25OH vitamin D deficiency, with adequate calciuria. Median phosphatemia was 1.0 mmol/L (0.79 to 1.4 mmol/L). Two patients (#05 and #06) had hypophosphatemia below 0.85 mmol/L without renal phosphate leakage, as indicated by normal tubular maximum reabsorption capacity of phosphate (TmP-GFR).Even if all patients received 25OH vitamin D replacement therapy, median 25OH vitamin D was 28 ng/ mL (15 to 76 ng/ mL). Seven out of 13 patients (54%) had 25OH vitamin D deficiency below 30 ng/mL. Median 1-25OH vitamin D was 43 pg/mL (23 to 106 pg/mL). Two patients had 1–25 OH vitamin D deficiency below 30 pg/mL, one of them (#08) having both 25OH and 1-25OH deficiency. Median PTH was 47.2 pg/mL . Six out of 13 patients (46%) had high PTH level above 50 pg/mL. Hyperparathyroidism was secondary to 25OH vitamin D deficiency in 2 patients and was associated with mGFR below 45 mL/min/1.73 mMedian magnesium in plasma was 0.83 mmol/L (from 0.68 to 0.96 mmol/L). Three out of 13 patients  had hypomagnesemia below 0.75 mmol/L. Patient #10 had a high urinary fractional excretion of the magnesium (11.3%), suggesting a renal loss of magnesium. Patients #06 and #07 had suitable fractional excretion of the magnesium.2. The other patients did not display any low molecular weight proteinuria.No patient had glycosuria. Only one patient, Patient #10, had a marked elevation of β2-microglobulinuria (506 μg/mmol), suggesting proximal tubulopathy. Interestingly, he also had the more severe CKD, defined by mGFR of 23.3 mL/min/1.73mTo conclude, no patient had complete tubular proximal syndrome. Only one patient (#10) had biological signs suggestive of incomplete proximal tubulopathy with both elevation of β2-microglobulinuria and renal loss of magnesium. Four out of 13 patients had isolated potassium loss related to a non-reabsorbable anion effect.No patient had microscopic hematuria or proteinuria above 500 mg/g. Median albumin to creatinine ratio was low 0.8 mg/mmol (0 to 15.4 mg/mmol). Three out of 13 patients  display albuminuria above 3 mg/mmol.2, 77% of patients had a mGFR below 90 mL/min/1.73 m2, more than half (54%) had at least moderate renal impairment (mGFR below 60 mL/min/1.73 m2), and one had severe renal impairment (mGFR below 15 mL/min/1.73 m2) [0 patients but is likely due to biased sampling of mut0 patients in our study. Indeed, 6 out of 9 MMA patients over 13-years of age followed in our hospital were not included in the present study because they previously received a liver and/or kidney graft , which reinforces this hypothesis. After renal transplantation, the number of decompensations per patient per year decreased and the protein intake significantly increased [CKD is a common complication of MMA and worsens long-term prognosis. We precisely studied the renal function of 13 adolescent and adult MMA patients who had not received a liver and/or kidney graft. Our study confirms the high prevalence of CKD in MMA patients: median mGFR was 56.5 mL/min/1.73 m1.73 m2) . Surprisr et al.  previousncreased . Whereasncreased .It has been suggested that CKD in MMA is the consequence of a tubular dysfunction. No case of glomerulopathy has been reported in the literature and our study supports this data. When performed, renal biopsy showed severe interstitial fibrosis and tubular atrophy , 24, 31,al. showed not only a disturbance of energy metabolism in glycolysis, mitochondrial respiratory chain and Krebs cycle but also increased reactive oxygen species formation [The hypothesis of a chronic tubulopathy is supported by experimental studies , 21, 22.ormation . Howeverormation , 25. In ormation , 35. Indormation . Moreoveormation . Lithiasormation . Gitelmaormation . Taking 2 with Schwartz formula and + 37 ± 22 mL/min/1.73 m2 with CKD-EPI [Furthermore, as expected, eGFR significantly overestimates renal function in MMA patients with low protein diet and reduced muscle mass: + 16 ± 15 mL/min/1.73 m CKD-EPI . Indeed,To conclude, CKD is a common complication of the MMA, which worsens long-term prognosis. Mild evidence of proximal tubular dysfunction was found in only one out of 13 patients, suggesting the existence of other mechanisms responsible for CKD during MMA. A better understanding of the mechanisms responsible for CKD in MMA is mandatory to improve the care of these patients.Additional file 1: Fig. S1. Methylmalonic acidemia is caused by a defect in the mitochondrial methylmalonyl-CoA mutase (MCM). MCM isomerises L-methylmalonyl-CoA into succinyl-CoA which enters the Krebs cycle. Its cofactor is adenosylcobalamin. The MUT, MMAA and MMAB genes respectively encode for MCM, CblA and CblB. MCM deficiencies are due to mutations in the MUT gene or to mutations in MMAA or MMAB. MCM deficiency results in accumulation of toxic metabolites such as 3-hydroxypropionic acid, methylcitric acid and most markedly, methylmalonic acid.Additional file 2: Fig. S2. Serum potassium was lower in patients with mut- phenotype. Medians ± interquartile ranges.


C3 glomerulopathy (C3G) is a recent disease classification that is characterized by the presence of glomerular deposits (composed of C3) in the absence of significant amounts of immunoglobulin and comprises dense deposit disease and C3 glomerulonephritis (C3GN). Most C3GN manifests as membranoproliferative, mesangial proliferative glomerulonephritis patterns via light microscopy. Pure membranous nephropathy (MN)-like glomerular lesions are rare manifestations of C3GN. Anti-neutrophil cytoplasmic antibodies (ANCAs) are also seldomly reported to be positive in C3GN. Herein, we report the case of a C3GN patient presenting with an MN-like glomerular pattern with ANCA positivity.A 68-year-old woman was admitted to a local hospital with elevated serum creatinine for two weeks. Laboratory tests showed a hemoglobin level of 85 g/L. Urinalysis was positive for 2 + protein and 360 RBCs/HPF. Blood biochemistry analysis revealed the following concentrations: albumin, 30.3 g/L; globulin, 46.2 g/L; blood urea nitrogen, 19.9 mmol/L; and serum creatinine, 234 µmol/L. The serum C3 level was 0.4950 g/L, and the serum C4 level was 0.1050 g/L. The direct Coombs test was positive. Serologic testing for ANCA revealed the presence of p-ANCA (1:10) by indirect immunofluorescence microscopy assay, as well as the presence of PR3 1.2  and MPO 3.5  by enzyme immunoassay. Renal biopsy sample pathology showed 2/6 cellular crescents and thickened glomerular basement membranes. Immunofluorescence testing revealed only diffuse, finely granular depositions of C3 along the glomerular capillary walls in frozen and paraffin-embedded tissue sections. Electron microscopy demonstrated the presence of subepithelial electron-dense deposits, similar to those that are observed in membranous nephropathy. Corticosteroid and cyclophosphamide were administered, with a subsequent improvement in renal function.We present the rare case of a patient with MN-like C3GN with ANCA positivity. C3GN with ANCA positivity may be represented by more crescents, severe renal dysfunction and more extrarenal manifestations. More cases are needed to elucidate the clinicopathologic features and optimal treatments of these patients. C3 glomerulopathy (C3G) is a recent disease classification that is characterized by the presence of glomerular deposits (composed of C3) in the absence of significant amounts of immunoglobulin (Ig) , 2. C3G Anti-neutrophil cytoplasmic antibodies (ANCAs) have been proven to cause pauci-immune necrotizing and crescentic GN and vasculitis . ANCA haA 68-year-old Chinese woman was admitted with elevated serum creatinine for two weeks. She noticed lower back pain and went to a local hospital two weeks prior. There were no symptoms of gross hematuria, foamy urine, frequent urination, urination urgency, urination pain, chills or fever. The laboratory tests revealed a serum creatinine concentration of 374 µmol/L and a urine protein level of 1.23 g/24 h. Therefore, she was transferred to our hospital. No special medication was previously taken by the patient.Physical examination showed prominent features of facial pallor. Her blood pressure was 143/76 mmHg. There were no palpable lymph nodes. The results of chest and abdominal exams were within normal limits, and mild edema of the lower extremities was noticed.9/L and a platelet count of 237 × 109/L. Urinalysis was positive for 2 + protein and 360 RBCs/HPF. The urinary protein/creatinine ratio was 0.482 g/mmol Cr. Fecal occult blood test results were negative. The blood biochemistry analysis revealed the following concentrations: albumin, 30.3 g/L; globulin, 46.2 g/L; blood urea nitrogen, 19.9 mmol/L; serum creatinine, 234 µmol/L; and uric acid, 254 µmol/L. Procalcitonin and C-reactive protein results were negative. The alpha fetoprotein, carbohydrate antigen-CA125, carbohydrate antigen-CA199 and carcinoembryonic antigen results were negative. Anti-nuclear antibody (ANA), anti-dsDNA and anti-Sm antibody results were negative. The serum C3 concentration was 0.4950 g/L , and serum C4 concentration was 0.1050 g/L . The direct Coombs test was positive. Serologic testing for ANCA revealed the presence of p-ANCA (1:10) by indirect immunofluorescence microscopy assay (IFA), and PR3 1.2  and MPO 3.5  by enzyme immunoassay (EIA). The serum IgG concentration was 36.0 g/L , whereas the serum IgG4 concentration was 1.19 g/L. Immunofixation electrophoresis did not reveal the presence of monoclonal immunoglobulin. Renal ultrasonography showed that the size of the right kidney was 97 × 46 × 45 mm3, whereas the left kidney measured 98 × 41 × 45 mm3.Laboratory tests showed a hemoglobin concentration of 85 g/L, a white blood cell count of 12.21 × 10A renal biopsy was performed. Light microscopy demonstrated the presence of four nearly normal glomeruli, except for the presence of two cellular crescents Fig.  a. The gShe was treated with 500 mg intravenous methylprednisolone for 3 days, followed by 30 mg oral prednisone per day, which was slowly tapered. Moreover, 400 mg intravenous cyclophosphamide was prescribed to be taken once a month. After treatment for six months, the serum creatinine level decreased to 178.0 µmol/L, and the serum albumin increased to 34.8 g/L Fig. . HemogloC3G is caused by the abnormal activation of complements due to the dysregulation of the alternative complement pathway. C3GN is a subtype of C3G, and it is characterized by dominant, nondense C3 deposits in the glomeruli, with an intensity of two or more orders of magnitude greater than any other Ig . C3GN can affect people of all ages. The average age of onset is approximately 30 years. The presentations of C3GN are heterogeneous and include proteinuria (mild to nephrotic syndrome), microhematuria, hypertension and chronic renal failure . MoreoveIt is noticeable that this patient exhibited serologic ANCA-positive results via IIF and ELISA. ANCA is strongly associated with small-vessel, pauci-immune vasculitis, such as granulomatosis with polyangiitis, microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis . ANCA teUntil now, only two C3GN cases with ANCA positivity have been reported. Chaudhuri reported the case of a 75-year-old male presenting with acute kidney injury with high levels of serum serine protease 3 antibodies (C-ANCAs) and rheumatoid factor (132 IU/mL), low serum C3 levels (57 mg/dL), severe anemia and thrombocytopenia . A renalAn optimal disease-directed treatment for C3G has yet to be determined. Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers can be used for their antiproteinuric effects. Based on the central role of complement abnormalities in the pathogenesis of C3G, anticomplement therapies seem to be potential treatments. Several case reports and small trials have supported the utility of anti-C5 therapy (eculizumab) in C3G patients, especially for patients with elevated soluble C5b-9 levels . ImmunosHerein, we presented the rare case of a patient with C3GN with predominant MN-like lesions, crescents and positive ANCA results. Patients with C3GN with ANCA positivity may present with more crescents, more severe renal dysfunction and more extrarenal manifestations. More cases are needed to elucidate the clinicopathologic characteristics and optimal treatment of these patients.


IMMH-010 is a prodrug of YPD-29B, which is a novel PD-L1 inhibitor. A specific and sensitive LC-MS/MS method with polarity switching was developed and validated for the simultaneous determination of IMMH-010 and YPD-29B in rat plasma, liver, brain, urine and fecal samples. Method validation was investigated to demonstrate the lower limit of quantification linearity, precision and accuracy, matrix effect and recovery, stability and dilution reliability for IMMH-010 and YPD-29B. This validated method was successfully applied to investigate the pharmacokinetics, tissue distribution, and excretion of IMMH-010 and YPD-29B in rats. After oral administration of IMMH-010 maleate to rats, IMMH-010 was rapidly and extensively converted to the active metabolite YPD-29B. The areas under the plasma concentration-time curve (AUC) of IMMH-010 and YPD-29B were proportional to the dose in the range of 10–100 mg/kg. IMMH-010 was primarily distributed in the adrenal gland, lymph nodes, heart, liver and spleen. YPD-29B was mainly observed in the liver, lymph, kidney, and lung. Approximately 28.81% of the IMMH-010 dose was recovered in the urine and feces within 72 h, including unchanged IMMH-010 (7.99%) and YPD-29B (20.82%). The results of this study may be useful as a reference for further development of IMMH-010 and PD-L1 inhibitors.Clinical Trial Registration: [https://clinicaltrials.gov/ct2/show/NCT04343859?term=IMMH-010&draw=2&rank=1], identifier [NCT04343859]." Cancer immunotherapy, which boosts the body’s immune system to induce an antitumor response, is a revolutionary anticancer strategy that has become a main focus of oncology research . One of 50 < 10−13 M (patent: CN109153670). IMMH-010 showed significant antitumor activity in various carcinoma xenograft models and entered phase I clinical trials with trial number NCT04343859 . Investigation of pharmacokinetic (PK) is an essential part of drug development, and a reliable analysis method is the basis of PK research. The objective of the present study was to explore a rapid, simple, sensitive and accurate LC-MS/MS method for the simultaneous determination of IMMH-010 and its active metabolite YPD-29B in rat biological matrices. This method was then successfully applied to study the ADME of IMMH-010 in rats and can provide useful information for clinical trials.IMMH-010, 2-[4-(2-bromo-biphenyl-3-ylmethoxy)-5-chloro-2-(pyridin-3-ylmethoxy)- benzylamino]-3-hydroxy-propionic acid isopropyl ester, can be hydrolyzed at the ester bond to produce the active metabolite YPD-29B. A pharmacological study demonstrated that YPD-29B could effectively block the binding of PD-1 and PD-L1 in a homogeneous time-resolved fluorescence (HTRF) protein-protein interaction assay, and the IC1), and IMM-H008B (IS2)  were provided by the Department of Chemosynthesis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. HPLC-grade methanol was obtained from Merck . Ammonium acetate was purchased from Dikma . Heparin sodium and sodium fluoride (NaF) were purchased from Sigma-Aldrich . Water was purified by a Milli-Q ultrapure water system .IMMH-010, IMMH-010 maleate, YPD-29B, IMM-H008BP  and an API 4000 mass spectrometer . Chromatographic separation of the 8 μL samples was conducted on a Zorbax SB-C18 column  in an oven at 35°C. The mobile phases were water with 1 mM ammonium acetate (phase A) and methanol (phase B) pumped at 0.3 ml/min. The elution conditions were as follows: 70% B, held for 2.8 min, increased to 100% B within 0.3 min, held for 3.7 min, returned to 70% B within 0.1 min and equilibrated for 5.1 min. The total chromatographic separation time was 12 min.1 and negative ion mode for YPD-29B and IS2. The MS parameters in positive mode were as follows: The ion spray voltage was maintained at 4500 V, the temperature at 550°C, the CAD gas at 10 psi, the CUR gas at 25 psi, gas1/gas2 at 50 psi, the declustering potential (DP) at 70 V for IMMH-010 and IS1, and the collision energy (CE) at 20 V for IMMH-010 and 23 V for IS1. The mass transition ion pairs were 641.3 → 494.2 for IMMH-010 and 664.4 → 517.5 for IS1. The MS parameters in negative mode were as follows: The ion spray voltage was maintained at -4500 V, the temperature at 550°C, the CAD gas at 12 psi, the CUR gas at 10 psi, gas 1/gas 2 at 80/60 psi, the DP at −110 V for YPD-29B and −103 V for IS1, and the CE at −45 V for YPD-29B and −53 V for IS2. The mass transition ion pairs were 597.1 → 154.8 for YPD-29B and 620.0 → 154.7 for IS2.Detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI) under the following conditions: positive ion mode for IMMH-010 and IS1, 3.20 mg/ml) and IMMH-008B  were prepared in DMSO. Gradient dilutions of the stock solutions of IMMH-010 and YPD-29B in acetonitrile were prepared for standard calibrators and quality control (QC). The IS stock solutions were diluted with acetonitrile and then mixed to prepare a working solution containing 1,580 ng/ml IS1 and 398 ng/ml IS2.Stock solutions of IMMH-010 (2.30 and 3.20 mg/ml), YPD-29 B (2.28 and 2.84 mg/ml), IMM-H008BP (ISPlasma: IMMH-010 is an ester-containing compound. Our preliminary results demonstrated that IMMH-010 was stable at 4°C in rat plasma containing 50 mM NaF (a broad-spectrum and potent esterase inhibitor). Therefore, heparin sodium and NaF were dissolved with 0.9% w/v sodium chloride to prepare a mixture of 0.5% heparin sodium and 500 mM NaF. All blood was withdrawn into EP tubes containing this mixture (1:9 v/v mixture:blood) followed by plasma separation. The plasma standards for IMMH-010 were prepared at 1, 2, 5, 10, 20, 80, 120 and 200 ng/ml, and those for YPD-29B were prepared at 2.5, 5, 10, 20, 50, 400, 600 and 1,000 ng/ml. QC samples were prepared at 1, 2, 40 and 160 ng/ml for IMMH-010 and 2.5, 5, 200 and 800 ng/ml for YPD-29B.Tissue homogenates, urine and feces: After rats were housed in separated metabolic cages to collect blank urine and feces, blank tissues (liver and brain) were collected, washed with normal saline, blotted dry with filter paper and added to normal saline (w:v = 1:3) for homogenization by a homogenizer. Tissue homogenate standards for IMMH-010 were prepared at 1, 2, 5, 10, 20, 80, 120, and 200 ng/ml, and those for YPD-29B were prepared at 2.5, 5, 10, 20, 50, 400, 600, and 1,000 ng/ml. QC samples were prepared at 1, 2, 40 and 160 ng/ml for IMMH-010 and 2.5, 5, 200, and 800 ng/ml for YPD-29B. The urine standards for IMMH-010 were prepared at 0.5, 1, 2.5, 10, 20, 40, and 60 ng/ml, and those for YPD-29B were prepared at 1, 3, 5, 20, 40, 50, and 80 ng/ml. QC samples were prepared at 0.5, 1, 5, and 50 ng/ml for IMMH-010 and 1, 3, 10, and 60 ng/ml for YPD-29B. Fecal standards for IMMH-010 were prepared at 1, 2, 5, 10, 20, 50, 60, and 100 ng/ml, and those for YPD-29B were prepared at 2.5, 5, 10, 20, 50, 400, 600, and 1,000 ng/ml. QC samples were prepared at 1, 2, 40, and 80 ng/ml for IMMH-010 and 2.5, 5, 200, and 800 ng/ml for YPD-29B.All samples before precipitation were maintained at 4°C. All blood was withdrawn into EP tubes containing a mixture of 0.5% heparin sodium and 500 mM NaF (1:9 v/v mixture:blood) and then centrifuged at 5,000 rpm for 10 min to collect plasma. The tissues were washed with normal saline, blotted dry with filter paper and homogenized with normal saline at 1:3 (w/v). Urine samples were directly diluted 10-fold in methanol. The air-dried fecal samples were homogenized with DMSO at 1:6 (w/v), followed by 100-fold dilution with methanol. For all the prepared samples, a 30-μL aliquot was added to 60 μL of a mixed IS solution. Then, the mixture was vortexed and centrifuged at 14,000 rpm for 5 min, and 8 μL of the supernatant was analyzed by LC-MS/MS.A selectivity assay was performed to identify whether there were any interferences in the six blank rat matrix samples  from separate sources that could influence the analysis of IMMH-010, YPD-29B and the IS. The peak area of interferences eluting at the retention times (RTs) of analytes and the IS was required to be less than 20% of the peak area in the lower limit of quantification (LLOQ) samples and less than 5% of the IS peak area.x2 (x = concentration) least squares regression.Linearity was evaluated from calibration curves with seven or eight concentrations for IMMH-010 and YPD-29 B. The linearity of a calibration curve was obtained by plotting the peak area ratio  vs. the nominal concentration using a linearly weighted 1/n = 6) on three separate days. The intra- and inter-day precision and accuracy were within ±15% for all QC samples and within ±20% for all LLOQ samples, indicating the established method have good reproducibility and accuracy.The relative standard deviation  and the relative error  were used to evaluate precision and accuracy, respectively. The intra- and inter-day precision and accuracy of the matrix were calculated with LLOQ samples and QC samples at low, intermediate and high levels each day . The MF is the ratio of the peak area of the analytes in a deproteinized blank matrix spiked with analytes to the peak area of the analytes in acetonitrile solution. The IS-normalized MF is the ratio of MFn = 6).The recovery was investigated by comparing the peak responses in blank matrix samples spiked with analyte to those in post-extracted samples spiked with analyte. Matrix effect and recovery were both evaluated at three concentration levels  (n = 5).For short-term and long-term stability, plasma samples were kept at 4°C for 4 h and −20°C for 3 months, respectively. To evaluate freeze-thaw stability, samples were frozen at −20°C for 24 h and thawed at 4°C for three cycles. For stability of analytical procedures, the prepared samples were stored in an autosampler at 10°C for 48 h before analysis. Stability assays of IMMH-010 and YPD-29B were performed at two QC levels (high and low QC)  with blank plasma, and the precision and accuracy were determined (n = 5).Female Sprague-Dawley rats  were kept in standard environmental conditions. Rats were fasted overnight with free access to water and randomly assigned to different groups before the experiments. All animal protocols were approved by the Institute Animal Care and Welfare Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College.IMMH-010 maleate was suspended in 0.5% sodium carboxymethyl cellulose. The rats were intragastrically (i.g.) administered IMMH-010 maleate at doses of 10, 30, and 100 mg/kg. Blood samples were withdrawn via the orbital plexus into heparinized tubes with NaF at 5, 15, and 30 min and 1, 2, 4, 6, 8, 12, 24, 36, and 48 h. Plasma was immediately prepared by centrifugation at 5,000 rpm for 10 min. All samples were stored at −20°C prior to analysis. The PK parameters of IMMH-010 and YPD-29B were calculated using the Linear Trapezoidal with Linear Interpolation method by WinNonlin software version 6.3 based on a noncompartmental model .The tissue distribution study was performed 15 min, 30 min, 4 h and 12 h after i. g. administration of 10 mg/kg IMMH-010 maleate. Blood was collected and centrifuged at 5,000 rpm for 10 min. The heart, liver, brain, spleen, lung, kidney, adrenal gland, thymus, and lymph were harvested immediately. The tissues were washed with normal saline and blotted dry with filter paper. All samples were stored at -20°C before analysis.After i. g. administration of IMMH-010 maleate (10 mg/kg), rats were housed in separate metabolite cages to collect their urine and feces: 0–12, 12–24, 24–36, 36–48, and 48–72 h for urine and 0–24, 24–48, and 48–72 h for feces. Fecal samples were dried in air. Urine samples were stored at −20°C prior to analysis.All data are expressed as the mean ± standard deviation (SD). The profiles of the mean blood concentration vs. time were plotted by GraphPad Prism 5 software .1) and IMM-H008B (IS2) were selected as the ISs of IMMH-010 and YPD-29B, respectively. In full-scan mode, the protonated molecular ion [M + H+2]+ was chosen as the parent ion for IMMH-010 and IS1, and the deprotonated form [M-H+2]- for YPD-29B and IS2. The predominant transitions for quantification were: m/z 641.3→494.2 for IMMH-010, m/z 664.4→517.5 for IS1, m/z 597.1→154.8 for YPD-29B and m/z 620.0→154.7 for IS2. Then, the DP, gas pressure and CE were optimized. The structures and product ion spectra of IMMH-010, YPD-29B and IS are presented in Although IMMH-010 and YPD-29 have similar chemical structures, IMMH-010, with a secondary amine group in its chain, exhibited a stronger signal intensity in positive ion mode, while YPD-29B, containing a carboxyl group, had a greater response in negative ion mode. Therefore, the positive-negative polarity switching mode was applied for the simultaneous determination of IMMH-010 and YPD29B. The structural analogs IMM-H008BP  on peak shape and analyte ionization were evaluated. Ammonium acetate (1 mM) improved the peak shape of YPD-29B, while the ionization of YPD-29B was suppressed when using 0.1% formic acid. Therefore, the mobile phase consisted of water and methanol with 1 mM ammonium acetate. Furthermore, when the initial concentration of methanol was 60%, the analytes could be separated from each other, and increasing the initial concentration of methanol resulted in increased resolution and decreased sensitivity. After comprehensively considering the resolution and sensitivity, a gradient starting with 70% methanol was employed, and all analytes achieved narrow peak widths and efficient separation, ensuring successful positive/negative polarity-switching analysis. Ultimately, in the first 5 min of the chromatographic run, YPD-29B and ISDue to the low ionization efficiency of YPD-29B in negative ion mode, efforts were made to optimize not only the HPLC separation conditions but also the sample preparation procedures, and liquid-liquid extraction, solid-phase extraction and various precipitation conditions were evaluated. When using Oasis HLB and Sep-Pak C18 Vac cartridges for solid-phase extraction, the recovery of IMMH-010 was extremely low. When using ether, ethyl acetate and dichloromethane for liquid-liquid extraction, the recovery of YPD-29B was too low to detect the two analytes simultaneously. The signals of IMMH-010 and YPD-29B after protein precipitation were decreased due to the matrix effect, but considering the advantages of protein precipitation , the conditions for protein precipitation using 20% trichloroacetic acid, methanol and acetonitrile were further optimized. The maximum sensitivity was achieved only when two volumes of acetonitrile were added. In addition, the response of YPD-29B was proportional to the injection volume below 8 μL, without peak broadening. Therefore, samples were precipitated with two volumes of acetonitrile, and 8 μL of supernatant was injected for quantification.1 were 5.80 and 5.76 min, respectively, and those of YPD-29B and IS2 were 3.95 and 3.80 min, respectively. There were no significant interferences from rat plasma at the RTs of the two analytes or ISs.r2) greater than 0.99. In the plasma, tissue and feces matrices, the LLOQs of IMMH-010 and YPD-29 B were 1 ng/ml and 2.5 ng/ml, and the ULOQs of IMMH-010 and YPD29B were 200 ng/ml (100 ng/ml for feces only) and 1,000 ng/ml, respectively. In the urine matrix, the LLOQs of IMMH-010 and YPD-29B were 0.5 ng/ml and 1 ng/ml, and the ULOQs of IMMH-010 and YPD29B were 60 ng/ml and 80 ng/ml, respectively. After injection of the ULOQ samples, blank samples were analyzed, and IMMH-010 and YPD-29B could not be found in the blank samples. Therefore, the carryover was negligible.As shown in The intra-day and inter-day precision and accuracy values of IMMH-010 and YPD-29B in plasma at the LLOQs and three QC levels are shown in The matrix effects and recoveries of IMMH-010 and YPD-29B in plasma samples at three QC levels are presented in The stability results are listed in Plasma samples with 800 ng/ml IMMH-010 and 4,000 ng/ml YPD-29B were diluted with blank plasma to yield 160 ng/ml IMMH-010 and 800 ng/ml YPD-29B. The precision and accuracy for the diluted samples are presented in max) at 0.5–1 h. The average plasma elimination half-life (t1/2β) of YPD-29B was 1.57–3.65 h. IMMH-010 was extensively converted to YPD-29B, with average AUC ratios (AUCYPD-29B/AUCIMMH-010) of 11.1, 13.4, and 12.5 for oral doses of 10, 30 and 100 mg/kg, respectively. From the Cmax-dose and AUC0-t-dose scatter diagrams in max of IMMH-010 and YPD-29B increased proportionally when the dose was increased . The mean residence time (MRT) of IMMH-010 and YPD-29B increased with the dose of IMMH-010, indicating that the pharmacokinetic properties of IMMH-010 exhibited saturation of eliminate in rat.The validated method was successfully applied to PK studies of IMMH-010 and YPD-29B in rats after a single i. g. administration of IMMH-010 maleate at 10, 30 and 100 mg/kg. The blood concentration-time profiles of the two analytes are presented in max values at 30 min postdose. IMMH-010 was distributed mainly in the adrenal gland, followed by the lymph, heart, liver and spleen. For YPD-29B, the highest T/P value was observed in the liver, followed by the lymph, kidney, and lung. The T/P values of the above tissues were greater than unity. The exposure of YPD-29B in the examined tissues was much higher than that of IMMH-010, with the exception of the heart, adrenal gland and brain. At 12 h, the concentrations of IMMH-010 and YPD-29B were below the LLOQ, indicating that the two analytes were quickly eliminated from tissues.After oral administration of 10 mg/kg IMMH-010 maleate to rats, the concentrations of IMMH-010 and YPD-29B in plasma and nine different tissues  and their tissue-to-plasma partition (T/P) values are shown in After oral administration of 10 mg/kg IMMH-010 maleate, the excretion of IMMH-010 in feces and urine is shown in A novel and simple polarity-switching LC-MS/MS method was developed and validated to simultaneously determine IMMH-010 and its active metabolite YPD-29B in rat plasma, liver, brain, urine, and fecal samples. This assay was successfully applied to investigate the pharmacokinetics, tissue distribution, and excretion of IMMH-010 and YPD-29B in rats. This is the first paper on the pharmacokinetics of a novel PD-L1 inhibitor. The results of this study may be useful as a reference for further development of IMMH-010 and PD-L1 inhibitors.


BG extract depolarized the pacemaker potentials of the ICC. Y25130 and RS39604 5-HT receptor antagonists could not inhibit the effect of BG extract on the pacemaker potentials of the ICC, whereas the 5-HT receptor antagonist SB269970 could. Pre-treatment with external Na+ (5 mM) or Ca2+-free solution inhibited the BG extract-induced depolarization of the ICC. With SB203580, PD98059, or c-jun NH2-terminal kinase II inhibitor pre-treatment, BG extract did not induce pacemaker potential depolarization. Moreover, the ITR values were increased by BG extract. Elevation of the ITR due to BG extract was related with increased protein expression of the 5-HT7 receptors. In addition, BG extract showed antioxidant activity. Collectively, these results highlight the ability of BG extract to regulate GI motility and the possibility of using it to develop GI motility modulators in the future. Moreover, BG showed immense potential as an antioxidant.Black garlic (BG) is a newly explored food stuff obtained via fermentation of raw, healthy garlic, especially in Asian countries. Interstitial cells of Cajal (ICC) are the pacemaker cells of gastrointestinal (GI) motility. The purpose of this study was to investigate the effects of BG extract on the pacemaker potentials of the ICC in the small intestines of mice and the possibility of controlling GI motility. The antioxidant activity of BG extract was also investigated. The whole-cell electrophysiological method was used to measure pacemaker potentials of the ICC  Allium sativum L.) has been used as a spice and traditional medicine for eons. Several studies have shown that garlic has beneficial effects on human health, such as anti-inflammatory, anti-cancer, lipid lowering, maintenance of blood pressure, and blood glucose regulation  are essential pacemaker cells that regulate the GI motility . A total of 2 kg of BG was extracted in 70% ethyl alcohol (20 L) for 6 h at 80°C and filtered through a Whatman No. 4 filter paper. After concentrating the solvents using rotary evaporation at 50°C, the yield was approximately 19.8% on a dry weight (w/w) basis.2 incubator.Small intestines of mice were excised and after removing the mucous membrane, cut it into pieces. The cells were dispersed in a solution containing various enzymes, including collagenase, and were cultured in smooth muscle growth medium  supplemented with a murine stem cell factor  in a 95% O2 2, glucose 10, MgCl2 1.2, and HEPES 10) was used to culture clusters of ICC. To examine the pacemaker activity, a solution comprising KCl 140, MgCl2 5, K2ATP 2.7, NaGTP 0.1, creatine phosphate disodium 2.5, HEPES 5, and EGTA 0.1 was added to the cultured clusters of the ICC using a pipette. Whole-cell configuration was performed using an Axopatch 200B amplifier .A physiological salt solution  was administered to healthy ICR mice after administration of BG extract into the stomach. After 30 min of Evans blue administration, the ITR of the mice was checked.We generated the GMD mouse models by using the acetic acid -induced peritoneal stimulation, as previously described , anti-5-HT4 receptor , anti-5-HT7 receptor , and anti-β-actin  antibodies. Other experiments are the same as in previous studies  membrane. After 1 h of incubation with a blocking buffer, PVDF membranes were probed with anti-5-HTForty-nine mice  from ICR were used for the ICC experiments. In addition, 39 mice  were used for the ITR experiments on heathy mice and mice with GI motility disease, whereas 10 mice  were used for the protein expression experiments. The ICC and ITR experiments were completed within 12 h of culturing, respectively. We experimented according to The Institutional Animal Care and Use Committee at Pusan National University approved . Also, animals were handled according to the Guide for the Care and Use of Laboratory Animals.A 0.2-mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution was prepared by dissolving DPPH reagent in EtOH. The prepared solution and BG extract were mixed in a 1:1 ratio and incubated for 30 min in a dark room. Absorbance at 517 nm was measured and DPPH radical scavenging activity was calculated using the following formula:2-terminal kinase (JNK) II inhibitor was purchased from Calbiochem . All other agents were purchased from Sigma-Aldrich.PD98059 and SB203580 were purchased from Tocris Bioscience (United Kingdom), whereas c-jun NHt-test. P values < 0.05 were considered as significant.Data are expressed as the mean ± standard error of the mean. Significant differences were evaluated using one-way analysis of variance (ANOVA) or the Student’s P < 0.001) at 1 mg/mL, 12.3 ± 0.9 mV  at 5 mg/mL, and 23.8 ± 1.4 mV  at 10 mg/mL  were identified in the murine small intestinal ICC , RS39604 (a 5-HT4 receptor antagonist), and SB269970 (a 5-HT7 receptor antagonist). Y25130 and RS39604 did not inhibit BG extract-induced responses (P < 0.0001) with SB269970 treatment  or Ca2+-free conditions. Pre-treatment with the external Na+ or Ca2+-free solution suppressed the pacemaker potentials and inhibited BG extract-induced responses (P < 0.0001) with Na+ solution and 3.4 ± 0.6 mV  with Ca2+-free solution  at 0.1 g/kg, and 66.0 ± 3.9%  at 1 g/kg (p < 0.0001), whereas for loperamide and BG extract, the ITR was 44.2 ± 3.8% , In mice, the ITR was 50.8 ± 1.9% n = 10; A. BG extt 1 g/kg A. LoperaP < 0.0001; P < 0.05), 45.3 ± 2.4% , and 60.5 ± 2.6% , respectively (P < 0.0001) and BG extract recovered this value to 35.6 ± 3.2%  (We generated the GMD mouse model. AA decreased the ITR (23.9 ± 1.5% (n = 10) vs. 51.9% ± 2.3% (n = 10) in normal mice; ectively B. Lopera 0.0001) B. These 3, 4, and 7 receptors were found in ICC  whereas that of 5-HT7 receptors increased by 228.9 ± 12.3%  after BG extract treatment  at 0.1 mg/mL, 46.4 ± 9.5%  at 1 mg/mL, and 55. 4 ± 11.4%  at 10 mg/mL; To investigate the antioxidant activity of BG extract, ROS scavenging activity was measured using the DPPH assay. BG extract significantly exhibited ROS scavenging activity  or Ca2+-free solution inhibited BG extract-induced pacemaker depolarization of ICC (in vivo (Garlic has been used as a medicinal ingredient for a long time (Jeong et al. n of ICC . BG extr(in vivo A. Moreov(in vivo B. Theref3 receptors, but Shahi et al. (3, 4, and 7 receptors. In addition, Wouters et al. (2B receptors regulate the growth of ICC. However, in this study, the 5-HT7 receptor antagonist SB269970 inhibited BG extract-induced responses, whereas the 5-HT3 and 5-HT4 receptor antagonists Y25130 and RS39604, respectively, did not. This shows that BG extract modulates pacemaker potentials due to the 5-HT7 receptors (7 receptors (7 receptors play a vital role in the regulation of GI motility by BG extract. 5-HT7 receptors are present in lymphoid tissues, smooth muscle cells, ICC, and neurons within the gut (Tonini et al. 7 receptors in GI motility regulation (Tonini et al. 7 receptors in GI motility and ICC in detail. In addition, MAPK signaling is also a major target for new treatments with GI motility disease (Ihara et al. ICC generate spontaneously active pacemaker potentials (Huizinga et al. i et al.  suggestes et al.  stated teceptors . In addieceptors . TherefoThe human body has a variety of complex antioxidant defense mechanisms to counter the harmful effects of free radicals or other oxidants (Alam et al. Recently, natural herbal medicine has been attracting increasing attention as an alternative medicine with few side effects Ekor . Since m7 receptors, extracellular Na+ and Ca2+ concentration regulation, and MAPK pathways (7 receptors. In addition, BG extract showed significant antioxidative effects. Therefore, BG might be a prokinetic agent that can cure or prevent GMD, and herbal medicine may become a very important strategy for the treatment of GI tract disorders.Collectively, the results from the present study showed that BG extract depolarized the pacemaker potentials of the ICC via the 5-HTpathways . Further