Edaravone dexborneol attenuates cognitive impairment in a rat model of vascular dementia by inhibiting hippocampal oxidative stress and inflammatory responses and modulating the NMDA receptor signaling pathway.

Exploring the intricate pathogenesis of Vascular Dementia (VD), there is a noted absence of potent treatments available in the current medical landscape. A new brain-protective medication developed in China, Edaravone dexboeol (EDB), has shown promise due to its antioxidant and anti-inflammatory properties, albeit with a need for additional research to elucidate its role and mechanisms in VD contexts. In a research setup, a VD model was established utilizing Sprague-Dawley (SD) rats, subjected to permanent bilateral typical carotid artery occlusion (2VO). Behavioral assessment of the rats was conducted using the Bederson test and pole climbing test, while cognitive abilities, particularly learning and memory, were evaluated via the novel object recognition test and the Morris water maze test. Ensuing, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), IL-1ß, IL-6, IL-4, and tumor necrosis factor-α (TNF-α) were determined through Enzyme-Linked Immunosorbent Assay (ELISA). Synaptic plasticity-related proteins, synaptophysin (SYP), post-synaptic density protein 95 (PSD-95), and N-methyl-D-aspartate (NMDA) receptor proteins (NR1, NR2A, NR2B) were investigated via Western blotting technique. The findings imply that EDB has the potential to ameliorate cognitive deficiencies, attributed to VD, by mitigating oxidative stress, dampening inflammatory responses, and modulating the NMDA receptor signaling pathway, furnishing new perspectives into EDB's mechanism and proposing potential avenues for therapeutic strategies in managing VD.

Characterization of a Novel M4 PAM PET Radioligand [11C]PF06885190 in Nonhuman Primates (NHP).

Muscarinic acetylcholine receptors (mAChR), including M4, draw attention as therapeutic targets for several neurodegenerative diseases including Alzheimer's disease (AD). PET imaging of M4 positive allosteric modulator (PAM) allows qualification of the distribution as well as the expression of this receptor under physiological conditions and thereby helps to assess the receptor occupancy (RO) of a drug candidate. In this study, our aims were (a) to synthesize a novel M4 PAM PET radioligand [11C]PF06885190 (b) to evaluate the brain distribution of [11C]PF06885190 in nonhuman primates (NHP) and (c) to analyze its radiometabolites in the blood plasma of NHP. Radiolabeling of [11C]PF06885190 was accomplished via N-methylation of the precursor. Six PET measurements were performed using two male cynomolgus monkeys, where three PET measurements were at baseline, two after pretreatment with a selective M4 PAM compound CVL-231 and one after pretreatment with donepezil. The total volume of distribution (VT) of [11C]PF06885190 was examined using Logan graphical analysis with arterial input function. Radiometabolites were analyzed in monkey blood plasma using gradient HPLC system. Radiolabeling of [11C]PF06885190 was successfully accomplished and the radioligand was found to be stable in the formulation, with radiochemical purity exceeding 99% 1 h after the end of the synthesis. [11C]PF06885190 was characterized in the cynomolgus monkey brain where a moderate brain uptake was found at the baseline condition. However, it showed fast wash-out as it dropped to half of the peak at around 10 min. Change of VT from baseline was around -10% after pretreatment with a M4 PAM, CVL-231. Radiometabolite studies showed relatively fast metabolism. Although sufficient brain uptake of [11C]PF06885190 was observed, these data suggest that [11C]PF06885190 might have too low specific binding in the NHP brain to be further applied in PET imaging.

Bivalirudin plus a high-dose infusion versus heparin monotherapy in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention: a randomised trial.

BACKGROUND: Previous randomised trials of bivalirudin versus heparin in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) have reported conflicting results, in part because of treatment with different pharmacological regimens. We designed a large-scale trial examining bivalirudin with a post-PCI high-dose infusion compared with heparin alone, the regimens that previous studies have shown to have the best balance of safety and efficacy. METHODS: BRIGHT-4 was an investigator-initiated, open-label, randomised controlled trial conducted at 87 clinical centres in 63 cities in China. Patients with STEMI undergoing primary PCI with radial artery access within 48 h of symptom onset who had not received previous fibrinolytic therapy, anticoagulants, or glycoprotein IIb/IIIa inhibitors were randomly assigned (1:1) to receive bivalirudin with a post-PCI high-dose infusion for 2-4 h or unfractionated heparin monotherapy. There was no masking. Glycoprotein IIb/IIIa inhibitor use was reserved for procedural thrombotic complications in both groups. The primary endpoint was a composite of all-cause mortality or Bleeding Academic Research Consortium (BARC) types 3-5 bleeding at 30 days. This trial is registered with ClinicalTrials.gov (NCT03822975), and is ongoing. FINDINGS: Between Feb 14, 2019, and April 7, 2022, a total of 6016 patients with STEMI undergoing primary PCI were randomly assigned to receive either bivalirudin plus a high-dose infusion after PCI (n=3009) or unfractionated heparin monotherapy (n=3007). Radial artery access was used in 5593 (93·1%) of 6008 patients. Compared with heparin monotherapy, bivalirudin reduced the 30-day rate of the primary endpoint (132 events [4·39%] in the heparin group vs 92 events [3·06%] in the bivalirudin group; difference, 1·33%, 95% CI 0·38-2·29%; hazard ratio [HR] 0·69, 95% CI 0·53-0·91; p=0·0070). All-cause mortality within 30 days occurred in 118 (3·92%) heparin-assigned patients and in 89 (2·96%) bivalirudin-assigned patients (HR 0·75; 95% CI 0·57-0·99; p=0·0420), and BARC types 3-5 bleeding occurred in 24 (0·80%) heparin-assigned patients and five (0·17%) bivalirudin-assigned patients (HR 0·21; 95% CI 0·08-0·54; p=0·0014). There were no significant differences in the 30-day rates of reinfarction, stroke, or ischaemia-driven target vessel revascularisation between the groups. Within 30 days, stent thrombosis occurred in 11 (0·37%) of bivalirudin-assigned patients and 33 (1·10%) of heparin-assigned patients (p=0·0015). INTERPRETATION: In patients with STEMI undergoing primary PCI predominantly with radial artery access, anticoagulation with bivalirudin plus a post-PCI high-dose infusion for 2-4 h significantly reduced the 30-day composite rate of all-cause mortality or BARC types 3-5 major bleeding compared with heparin monotherapy. FUNDING: Chinese Society of Cardiology Foundation (CSCF2019A01), and a research grant from Jiangsu Hengrui Pharmaceuticals.

Target occupancy study and whole-body dosimetry with a MAGL PET ligand [11C]PF-06809247 in non-human primates.

BACKGROUND: Monoacylglycerol lipase (MAGL) is a key serine hydrolase which terminates endocannabinoid signaling and regulates arachidonic acid driven inflammatory responses within the central nervous system. To develop [11C]PF-06809247 into a clinically usable MAGL positron emission tomography (PET) radioligand, we assessed the occupancy of MAGL by an inhibitor in the non-human primate (NHP) brain. Additionally, we measured the whole-body distribution of [11C]PF-06809247 in NHP and estimated human effective radiation doses. METHODS: Seven cynomolgus monkeys were enrolled for brain PET measurements. Two PET measurements along with arterial blood sampling were performed in each NHP: one baseline and one pretreatment condition with intravenous administration of PF-06818883, a pro-drug of a selective MAGL inhibitor (total of seven doses between 0.01 and 1.27 mg/kg). Kinetic parameters K1, k2 and k3 were estimated by a two tissue compartment (2TC) model using metabolite corrected plasma radioactivity as the input function. k4 was set as 0 according to the irreversible binding of [11C]PF-06809247. Ki by 2TC and Patlak analysis were calculated as the influx constant. The target occupancy was calculated using Ki at baseline and pretreatment conditions. Two cynomolgus monkeys were enrolled for whole-body PET measurements. Estimates of the absorbed radiation dose in humans were calculated with OLINDA/EXM 1.1 using the adult male reference model. RESULTS: Radioactivity retention was decreased in all brain regions following pretreatment with PF-06818883. Occupancy was measured as 25.4-100.5% in a dose dependent manner. Whole-body PET showed high radioactivity uptake values in the liver, small intestine, kidney, and brain. The effective dose of [11C]PF-06809247 was calculated as 4.3 µSv/MBq. CONCLUSIONS: [11C]PF-06809247 is a promising PET ligand for further studies of MAGL in the human brain.

Development of 11C-Labeled ASEM Analogues for the Detection of Neuronal Nicotinic Acetylcholine Receptors (α7-nAChR).

The homo-pentameric alpha 7 receptor is one of the major types of neuronal nicotinic acetylcholine receptors (α7-nAChRs) related to cognition, memory formation, and attention processing. The mapping of α7-nAChRs by PET pulls a lot of attention to realize the mechanism and development of CNS diseases such as AD, PD, and schizophrenia. Several PET radioligands have been explored for the detection of the α7-nAChR. 18F-ASEM is the most functional for in vivo quantification of α7-nAChRs in the human brain. The first aim of this study was to initially use results from in silico and machine learning techniques to prescreen and predict the binding energy and other properties of ASEM analogues and to interpret these properties in terms of atomic structures using 18F-ASEM as a lead structure, and second, to label some selected candidates with carbon-11/hydrogen-3 (11C/3H) and to evaluate the binding properties in vitro and in vivo using the labeled candidates. In silico predictions are obtained from perturbation free-energy calculations preceded by molecular docking, molecular dynamics, and metadynamics simulations. Machine learning techniques have been applied for the BBB and P-gp-binding properties. Six analogues of ASEM were labeled with 11C, and three of them were additionally labeled with 3H. Binding properties were further evaluated using autoradiography (ARG) and PET measurements in non-human primates (NHPs). Radiometabolites were measured in NHP plasma. All six compounds were successfully synthesized. Evaluation with ARG showed that 11C-Kln83 was preferably binding to the α7-nAChR. Competition studies showed that 80% of the total binding was displaced. Further ARG studies using 3H-KIn-83 replicated the preliminary results. In the NHP PET study, the distribution pattern of 11C-KIn-83 was similar to other α7 nAChR PET tracers. The brain uptake was relatively low and increased by the administration of tariquidar, indicating a substrate of P-gp. The ASEM blocking study showed that 11C-KIn-83 specifically binds to α7 nAChRs. Preliminary in vitro evaluation of KIn-83 by ARG with both 11C and 3H and in vivo evaluation in NHP showed favorable properties for selectively imaging α7-nAChRs, despite a relatively low brain uptake.

Synthesis and Autoradiography of Novel F-18 Labeled Reversible Radioligands for Detection of Monoamine Oxidase B.

Monoamine oxidase B (MAO-B) is an important enzyme regulating the levels of monoaminergic neurotransmitters. Selective MAO-B inhibitors have been labeled with carbon-11 or fluorine-18 to visualize the localization of MAO-B in vivo by positron emission tomography (PET) and thereby have been useful for studying neurodegenerative diseases. The aim of this study was to develop promising fluorine-18 labeled reversible MAO-B PET radioligands and their biological evaluation in vitro by autoradiography. Radiolabeling was achieved by classical one-step fluorine-18 nucleophilic substitution reaction. The stability and radiochemical yield was analyzed with HPLC. All five fluorine-18 labeled compounds were tested in human whole hemisphere autoradiography experiments. Five compounds (GEH200439, GEH200448, GEH200449, GEH200431A, and GEH200431B) were successfully radiolabeled with fluorine-18, and the incorporation yield of the fluorination reactions varied from 10 to 45% depending on the compound. The radiochemical purity was higher than 99% for all at the end of synthesis. Radioligands were found to be stable, with a radiochemical purity of >99% in a sterile phosphate buffered saline (pH = 7.4) over the duration of the study. The ARG binding density of only 18F-GEH200449 was consistent with known MAO-B expression in the human brain. Radiolabeling of five new fluorine-18 MAO-B reversible inhibitors was successfully accomplished. Compound 18F-GEH200449 binds specifically to MAO-B in vitro postmortem brain and could be a potential candidate for in vivo PET investigation.

Identification and in vitro characterization of C05-01, a PBB3 derivative with improved affinity for alpha-synuclein.

The neuropathological hallmark of Parkinsons disease, multiple system atrophy and dementia with Lewy bodies is the accumulation of α-synuclein. The development of an imaging biomarker for α-synuclein is an unmet need. To date, no selective α-synuclein imaging agent has been identified, though initial studies suggest that the tau tracer [11C]PBB3 displays some degree of binding to α-synuclein. In this study, a series of compounds derived from the PBB3 scaffold were examined using fluorescence imaging and tissue microarrays (TMAs) derived from brain samples with different proteinopathies. One compound, C05-01, was selected based on its higher fluorescence signal associated with Lewy body aggregates compared with other PBB3 analogues. In vitro binding assays using human brain homogenates and recombinant fibrils indicated that C05-01 had higher affinity for α-synuclein (KD/Ki 25 nM for fibrils, Ki 3.5 nM for brain homogenates) as compared with PBB3 (KD 58 nM). In autoradiography (ARG) studies using fresh frozen human tissue and TMAs, [3H]C05-01 displayed specific binding in cases with α-synuclein pathology. C05-01 is the first PBB3 analogue developed as a potential compound targeting α-synuclein. Despite improved affinity for α-synuclein, C05-01 showed specific binding in AD tissue with Amyloid ß and tau pathology, as well as relatively high non-specific and off-target binding. Additional efforts are needed to optimize the pharmacological and physicochemical properties of this series of compounds as ligands for α-synuclein. This study also showed that the construction of TMAs from different proteinopathies provides a tool for evaluation of fluorescent or radiolabelled compounds binding to misfolded proteins.

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Impacts of CYP2C19 Polymorphism and Clopidogrel Dosing on in-Stent Restenosis: A Retrospective Cohort Study in Chinese Patients.

OBJECTIVE: This retrospective cohort study is to analyze the impacts of CYP2C19 polymorphism and clopidogrel dosing on in-stent restenosis (ISR) after coronary stenting. METHODS: Totally, 111 patients were included, who underwent percutaneous coronary intervention (PCI) with drug-eluting stent. Patients received clopidogrel treatment after the intervention on the background treatment with aspirin, based on the genotypes: 75 mg clopidogrel once each day for subjects without CYP2C19 loss-of-function (LOF) alleles (n=51; EM), 75 mg clopidogrel once each day (n=27; IM75) or twice each day (n=33; IM150) for subjects with one CYP2C19 LOF allele. ISR at 3-18 months after coronary stenting was assessed. RESULTS: ISR rate was significantly higher in the IM75 group (40.7%) than the EM group (11.8%). ISR rate in the IM150 group was lower than the IM75 group (6.1% vs 40.7%), and comparable to that in the EM group. Multivariate logistic regression showed that both CYP2C19 genotype and clopidogrel dosing were associated with the risk of ISR after adjusting the relevant confounding factors. ISR risk was higher in the IM patients than the EM patients. Patients with clopidogrel dose of 75 mg once each day had significantly higher risk of ISR than those with the dose of 75 mg twice each day. CONCLUSION: Increased dose of clopidogrel may reduce the risk of ISR after PCI in CYP2C19 LOF allele(s) carriers. The presence of CYP2C19 LOF allele(s) increases the risk of ISR after stenting, which could be counteracted by the increased dose of clopidogrel.

Identification and Development of an Irreversible Monoacylglycerol Lipase (MAGL) Positron Emission Tomography (PET) Radioligand with High Specificity.

Monoacylglycerol lipase (MAGL), a serine hydrolase extensively expressed throughout the brain, serves as a key gatekeeper regulating the tone of endocannabinoid signaling. Preclinically, inhibition of MAGL is known to provide therapeutic benefits for a number of neurological disorders. The availability of a MAGL-specific positron emission tomography (PET) ligand would considerably facilitate the development and clinical characterization of MAGL inhibitors via noninvasive and quantitative PET imaging. Herein, we report the identification of the potent and selective irreversible MAGL inhibitor 7 (PF-06809247) as a suitable radioligand lead, which upon radiolabeling was found to exhibit a high level of MAGL specificity; this enabled cross-species measurement of MAGL brain expression (Bmax), assessment of in vivo binding in the rat, and nonhuman primate PET imaging.

Characterization of [11C]PXT012253 as a PET Radioligand for mGlu4 Allosteric Modulators in Nonhuman Primates.

PURPOSE: Modulation of presynaptic metabotropic glutamate receptor 4 (mGlu4) by an allosteric ligand has been proposed as a promising therapeutic target in Parkinson's disease and levodopa-induced dyskinesia. A positron emission tomography (PET) ligand for an allosteric site of mGlu4 may provide evidence that a clinical drug candidate reaches and binds the target. A carbon-11-labeled PET radioligand binding an allosteric site of mGlu4, [11C]PXT012253, has been recently developed. Here, we describe the detailed characterization of this novel radiolabeled mGlu4 ligand in nonhuman primates. PROCEDURES: [11C]PXT012253 binding in the brain of cynomolgus monkeys, under the baseline and blocking conditions with the structurally different mGlu4 allosteric ligand PXT002331, currently in clinical trials for Parkinson's disease, was quantified with compartment and graphical modeling approaches using a radiometabolite-corrected plasma input function. Whole-body biodistribution of [11C]PXT012253 was then assessed using PET/x-ray computed tomography to estimate the human effective doses of [11C]PXT012253 for further clinical studies. RESULTS: [11C]PXT012253 displayed binding in mGlu4-expressing regions in the brain of cynomolgus monkeys. Brain regional time-activity curves of [11C]PXT012253 were well described in the two-tissue compartment model (2TC). Total distribution volume was stably estimated using Logan plot and multilinear analysis (MA1) although 2TC showed unstable values in some cases. Competition with PXT002331 showed high specific binding in the total distribution volume. Whole-body PET showed high accumulation of [11C]PXT012253 in the liver, kidney, heart, and brain in the initial phase. The radioligand was excreted through both the gastrointestinal and the urinary tracts. Effective dose of [11C]PXT012253 was estimated to be 0.0042 mSv/MBq. CONCLUSIONS: [11C]PXT012253 was shown to be a promising PET radioligand for mGlu4 allosteric modulators in the monkey brain. MA1 would be the choice of quantitative method. Further development of [11C]PXT012253 in human subjects is warranted.

The development of a GPR44 targeting radioligand [11C]AZ12204657 for in vivo assessment of beta cell mass.

BACKGROUND: The G-protein-coupled receptor 44 (GPR44) is a beta cell-restricted target that may serve as a marker for beta cell mass (BCM) given the development of a suitable PET ligand. METHODS: The binding characteristics of the selected candidate, AZ12204657, at human GPR44 were determined using in vitro ligand binding assays. AZ12204657 was radiolabeled using 11C- or 3H-labeled methyl iodide ([11C/3H]CH3I) in one step, and the conversion of [11C/3H]CH3I to the radiolabeled product [11C/3H]AZ12204657 was quantitative. The specificity of radioligand binding to GPR44 and the selectivity for beta cells were evaluated by in vitro binding studies on pancreatic sections from human and non-human primates as well as on homogenates from endocrine and exocrine pancreatic compartments. RESULTS: The radiochemical purity of the resulting radioligand [11C]AZ12204657 was > 98%, with high molar activity (MA), 1351 ± 575 GBq/µmol (n = 18). The radiochemical purity of [3H]AZ12204657 was > 99% with MA of 2 GBq/µmol. Pancreatic binding of [11C/3H]AZ12204657 was co-localized with insulin-positive islets of Langerhans in non-diabetic individuals and individuals with type 2 diabetes (T2D). The binding of [11C]AZ12204657 to GPR44 was > 10 times higher in islet homogenates compared to exocrine homogenates. In human islets of Langerhans GPR44 was co-expressed with insulin, but not glucagon as assessed by co-staining and confocal microscopy. CONCLUSION: We radiolabeled [11C]AZ12204657, a potential PET radioligand for the beta cell-restricted protein GPR44. In vitro evaluation demonstrated that [3H]AZ12204657 and [11C]AZ12204657 selectively target pancreatic beta cells. [11C]AZ12204657 has promising properties as a marker for human BCM.

A Nonhuman Primate PET Study: Measurement of Brain PDE4 Occupancy by Roflumilast Using (R)-[11C]Rolipram.

PURPOSE: Phosphodiesterase 4 (PDE4) inhibition in the brain has been reported to improve cognitive function in animal models. Therefore, PDE4 inhibitors are one of key targets potential for drug development. Investigation of brain PDE4 occupancy would help to understand the effects of PDE4 inhibition to cognitive functions. Roflumilast is a selective phosphodiesterase type 4 (PDE4) inhibitor used clinically for severe chronic obstructive pulmonary disease, but the effects to the brain have not been well investigated. In this study, we aimed to investigate whether roflumilast entered the brain and occupied PDE4 in nonhuman primates. PROCEDURES: Positron emission tomography (PET) measurements with (R)-[11C]rolipram were performed at baseline and after intravenous (i.v.) administration of roflumilast (3.6 to 200 µg/kg) in three female rhesus monkeys. Arterial blood samples were taken to obtain the input function. Protein binding was measured to obtain the free fraction (fp) of the radioligand. Total distribution volume (VT) and VT/fp were calculated as outcome measures from two tissue compartment model. Lassen plot approach was taken to estimate the target occupancy. RESULTS: The brain uptake of (R)-[11C]rolipram decreased after roflumilast administration. PDE 4 occupancy by roflumilast showed dose- and plasma concentration-dependent increase, although PDE4 occupancy did not reach 50 % even after the administration of up to 200 µg/kg of roflumilast, regardless of outcome measures, VT or VT/fp. CONCLUSIONS: This PET study showed that the brain PDE4 binding was blocked to a certain extent after i.v. administration of clinical relevant doses of roflumilast in nonhuman primates. Further clinical PET evaluation is needed to understand the relationship between PDE4 inhibition and potential improvement of cognitive function in human subjects.

Comparative binding properties of the tau PET tracers THK5117, THK5351, PBB3, and T807 in postmortem Alzheimer brains.

BACKGROUND: The aim of this study was to compare the binding properties of several tau positron emission tomography tracers-THK5117, THK5351, T807 (also known as AV1451; flortaucipir), and PBB3-head to head in the same human brain tissue. METHODS: Binding assays were performed to compare the regional distribution of 3H-THK5117 and 3H-THK5351 in postmortem tissue from three Alzheimer's disease (AD) cases and three control subjects in frontal and temporal cortices as well as in the hippocampus. Competition binding assays between THK5351, THK5117, PBB3, and T807, as well as off-target binding of THK5117 and T807 toward monoamine oxidase B (MAO-B), were performed using binding assays in brain homogenates and autoradiography of three AD cases. RESULTS: Regional binding of 3H-THK5117 and 3H-THK5351 was similar, except in the temporal cortex, which showed higher 3H-THK5117 binding. Saturation studies demonstrated two binding sites for 3H-THK5351 (K d1 = 5.6 nM, Bmax = 76 pmol/g; K d2 = 1 nM, Bmax = 40 pmol/g). Competition studies in the hippocampus between 3H-THK5351 and unlabeled THK5351, THK5117, and T807 revealed super-high-affinity sites for all three tracers (THK5351 K i = 0.1 pM; THK5117 K i = 0.3 pM; T807 K i = 0.2 pM) and an additional high-affinity site (THK5351 K i = 16 nM; THK5117 K i = 20 nM; T807 K i = 78nM). 18F-T807, 11C-THK5351, and 11C-PBB3 autoradiography of large frozen sections from three AD brains showed similar regional binding for the three tracers, with lower binding intensity for 11C-PBB3. Unlabeled THK5351 and T807 displaced 11C-THK5351 to a similar extent and a lower extent, respectively, compared with 11C-PBB3. Competition with the MAO-B inhibitor 3H-L-deprenyl was observed for THK5117 and T807 in the hippocampus (THK5117 K i = 286 nM; T807 K i = 227 nM) and the putamen (THK5117 K i = 148 nM; T807 K i = 135 nM). 3H-THK5351 binding was displaced using autoradiography competition with unlabeled THK5351 and T807 in cortical areas by 70-80% and 60-77%, respectively, in the basal ganglia, whereas unlabeled deprenyl displaced 3H-THK5351 binding by 40% in the frontal cortex and 50% in the basal ganglia. CONCLUSIONS: THK5351, THK5117, and T807 seem to target similar binding sites, but with different affinities, whereas PBB3 seems to target its own binding site. Both THK5117 and T807 demonstrated off-target binding in the hippocampus and putamen with a ten times lower binding affinity to the MAO-B inhibitor deprenyl compared with 3H-THK5351.

Development of [11C]/[3H]THK-5351 - A potential novel carbon-11 tau imaging PET radioligand.

INTRODUCTION: Due to the rise in the number of patients with dementia the imperative for finding new diagnostic and treatment options becomes ever more pressing. While significant progress has been made in PET imaging of Aß aggregates both in vitro and in vivo, options for imaging tau protein aggregates selectively are still limited. Based on the work previously published by researchers from the Tohoku University, Japan, that resulted in the development of [18F]THK-5351, we have undertaken an effort to develop a carbon-11 version of the identical structure - [11C]THK-5351. In parallel, THK-5351 was also labeled with tritium ([3H]THK-5351) for use in in vitro autoradiography (ARG). METHODS: The carbon-11 labeling was performed starting with di-protected enantiomeric pure precursor - tert-butyl 5-(6-((2S)-3-fluoro-2-(tetrahydro-2H-pyran-2-yloxy)propoxy)quinolin-2-yl)pyridin-2-yl carbamate, which was reacted with [11C]MeI, using DMF as the solvent and NaH as base, followed by deprotection with trifluoroacetic acid/water mixture, resulting in enantiomerically pure carbon-11 radioligand, [11C]THK-5351 - (S)-1-fluoro-3-(2-(6-([11C]methylamino)pyridin-3-yl)quinolin-6-yloxy)propan-2-ol. Tritium labeling and purification of [3H]THK-5351 were undertaken using similar approach, resulting in [3H]THK-5351 with RCP >99.8% and specific radioactivity of 1.3GBq/µmol. RESULTS: [11C]THK-5351 was produced in good yield (1900±355MBq), specific radioactivity (SRA) (361±119GBq/µmol at EOS+20min) and radiochemical purity (RCP) (>99.8%), with enantiomeric purity of 98.7%. [3H]THK-5351 was evaluated for ARG of tau binding in post-mortem human brain tissue using cortical sections from one AD patient and one control subject. [3H]THK-5351 binding density was higher in the AD patient compared to the control subject, the binding was displaced by unlabeled THK-5351 confirming specific [3H]THK-5351 binding.

Amyloid binding properties of curcumin analogues in Alzheimer's disease postmortem brain tissue.

The presence of ß-amyloid (Aß) containing plaques in the brain is a hallmark of Alzheimer's disease (AD) and serves as a biomarker for confirmation of diagnosis postmortem. Early diagnosis is of great importance for optimal treatment and for monitoring disease progression in the brain. Highly specific and sensitive biomarkers are thus greatly needed to assess therapeutic efficacy, not only clinically, but also in terms of clearance of histopathological lesions and decelerated neurodegeneration. The objective of the present study was to give more insight into the binding of curcumin analogues, curcuminoids, to Aß containing plaques in postmortem tissue from AD patients. In vitro autoradiography was utilized to explore affinity and displacement of the curcuminoids; curcumin, demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC) and dimethoxycurcumin (DIMC). We found that BDMC had the highest affinity for Aß containing plaques in cortical AD brain tissue in comparison to other curcuminoids. Subsequently, [(3)H]BDMC showed significantly higher specific binding in cortical AD brain tissue compared to control subjects. These findings suggest that curcumin analogues, especially BDMC, may serve as a potential radioligands for Aß plaque neuroimaging.

[11C]MADAM Used as a Model for Understanding the Radiometabolism of Diphenyl Sulfide Radioligands for Positron Emission Tomography (PET).

In quantitative PET measurements, the analysis of radiometabolites in plasma is essential for determining the exact arterial input function. Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT) and it is therefore important to investigate their radiometabolism. We have chosen to explore the radiometabolic profile of [11C]MADAM, one of these radioligands widely used for in vivo PET-SERT studies. The metabolism of [11C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the radiometabolic rate of the radioligand [11C]MADAM in vitro and in vivo was examined by radio-HPLC. RLM and HLM incubations were carried out at two different carrier concentrations of 1 and 10 µM. Urine samples after perfusion of [11C]MADAM/MADAM in rats were also analysed by radio-HPLC. Analysis by UHPLC/Q-ToF-MS identified the metabolites produced in vitro to be results of N-demethylation, S-oxidation and benzylic hydroxylation. The presence of carrier greatly affected the radiometabolism rate of [11C]MADAM in both RLM/HLM experiments and in vivo rat studies. The good concordance between the results predicted by RLM and HLM experiments and the in vivo data obtained in rat studies indicate that the kinetics of the radiometabolism of the radioligand [11C]MADAM is dose-dependent. This issue needs to be addressed when the diarylsulfide class of compounds are used in PET quantifications of SERT.

Safflower extract inhibiting apoptosis by inducing autophagy in myocardium derived H9C2 cell.

The Heart failure (HF) is considered as the end-stage of various heart disease and associated with high mortality globally. Progressive loss of cardiac myocytes via apoptosis is considered as the most important factor for HF pathology. In this study, we demonstrated that Safflower extract was able to inhibitthe apoptosis inducted by Angiotensin II (AngII) in a ratmyocardium derived cell line H9C2. Further examination of LC-3II conversion and autophagosome formation suggested Safflower extract induced autophagy in treated cell. Inhibition of Safflower extract induced autophagy by 3-methyladenine (3MA) abolished anti-apoptotic function of Safflower extract, while application of autophagy stimulator Rapamycin in H9C2 inhibited apoptosis as well. Moreover, treatment of H9C2 cell with Safflower extract also inhibited expression of pro-apoptotic genes BAD and Bax. In conclusion, our data indicated that Safflower extract inhibit apoptosis via inducing autophagy in myocardium cell and demonstrated the potential as novel therapeutic drug for Heart failure.

Decrease of mGluR5 receptor density goes parallel with changes in enkephalin and substance P immunoreactivity in Huntington's disease: a preliminary investigation in the postmortem human brain.

Group 1 metabotropic glutamate subtype 5 receptors (mGluR5) contribute to the control of motor behavior by regulating the balance between excitation and inhibition of outputs in the basal ganglia. The density of these receptors is increased in patients with Parkinson's disease and motor complications. We hypothesized that similar changes may occur in Huntington's disease (HD) and aimed at testing this hypothesis in a preliminary experimental series in postmortem human brain material obtained from HD patients. Using autoradiography, we analyzed mGluR5 density in the putamen, caudate nucleus and cerebellum (control region) in postmortem tissue samples from three patients with HD and three controls with two mGluR5-specific radioligands ([(3)H]ABP688 and [(11)C]ABP688). The density of enkephalin (Enk)- or substance P (SP)-containing neurons was assessed using immunohistochemical and cell-counting methods. [(3)H]ABP688 binding in HD was reduced in the caudate (-70.4 %, P < 0.001), in the putamen (-33.3 %, P = 0.053), and in the cerebellum (-8.79 %, P = 0.930) vs controls. Results with [(11)C]ABP688 were similar; there was good correlation between [(11)C]ABP688 and [(3)H]ABP688 binding ratios. Total cell density was similar in all three brain regions in HD patients and controls. Neuronal density was 69 % lower in the caudate (P = 0.002) and 64 % lower in the putamen (P < 0.001) of HD patients vs controls. Both direct and indirect pathways were affected, with ≥ 90 % decrease in the density of Enk- and SP-containing neurons in the caudate and putamen of HD patients vs controls (P < 0.001). In contrast to earlier observations in PD, in HD, compared to controls, the mGluR5 density was significantly lower in the caudate nucleus. The decrease in neuronal density suggests that neuronal loss was largely responsible for the observed decrease in mGluR5.