Exploring the in vitro stability of insulin degrading enzyme as a potential biomarker for neurocognitive disorders and Alzheimer's disease risk.

Insulin degrading enzyme (IDE) plays a critical role in degrading insulin and beta-forming proteins, implicating its significance as a biomarker in metabolic dysfunction and neurocognitive disorders, including Alzheimer's disease (AD). Understanding the impact of pre-analytic conditions of in vitro IDE levels is imperative for reliable biomarker assessment. This study explored the influence of freeze-thaw cycles, storage temperature, and storage time on IDE levels in human serum. Serum samples from seven healthy volunteers were subjected to various storage conditions, including refrigeration (4 °C) and freezing (-20 °C and -80 °C) for 24 h and six months, with differing freeze-thaw cycles. In vitro IDE levels were measured at 24 h and after 6 months using ELISA. Results indicate that while short-term storage at either -20 °C or -80 °C yielded similar IDE levels, prolonged storage and multiple freeze-thaw cycles significantly impacted IDE stability, with colder temperatures exhibiting better preservation. Although further research with larger cohorts and longer storage time is warranted to establish clinical significance, our study suggests preferential use of unthawed samples or consistent freeze-thaw conditions for accurate IDE assessment. Thus, optimizing sample storage conditions is paramount for reliable IDE biomarker analysis in clinical and research settings.

Correlations between insulin-degrading enzyme and metabolic markers in patients diagnosed with type 2 diabetes, Alzheimer's disease, and healthy controls: a comparative study.

PURPOSE: This study aimed to explore correlations between insulin-degrading enzyme (IDE) and markers of metabolic function in a group of patients diagnosed with type 2 diabetes mellitus (T2DM) or Alzheimer's disease (AD) and metabolically healthy volunteers. METHOD: We included 120 individuals (47 with T2DM, 9 with AD, and 64 healthy controls). Serum levels of IDE were measured with commercial kits for ELISA. Differences in IDE levels between groups were analyzed with non-parametric ANCOVA, and correlations were analyzed with Spearman's rank correlations. We also investigated the influence of age, sex, and the use of insulin on the correlation using a non-parametric version of partial correlation. RESULTS: Patients diagnosed with T2DM had higher IDE levels than patients diagnosed with AD and healthy controls after adjustment for age and sex. IDE was increasingly associated with body mass index (BMI), fasting blood glucose, C-peptide, hemoglobin A1c (HbA1c), insulin resistance, and triglycerides. In stratified analyses, we found a decreasing partial correlation between IDE and HbA1c in patients diagnosed with AD and a decreasing partial correlation between IDE and C-peptide in healthy controls. In patients diagnosed with T2DM, we found no partial correlations. CONCLUSION: These results indicate that IDE is essential in metabolic function and might reflect metabolic status, although it is not yet a biomarker that can be utilized in clinical practice. Further research on IDE in human blood may provide crucial insights into the full function of the enzyme.

Microsome Mediated in Vitro Metabolism: A Convenient Method for the Preparation of the PET Radioligand Metabolite [18F]FE-PE2I-OH for Translational Dopamine Transporter Imaging.

Undesired radiometabolites can be detrimental to the development of positron emission tomography (PET) radioligands. Methods for quantifying radioligand metabolites in brain tissue include ex vivo studies in small animals or labeling and imaging of the radiometabolite(s) of interest. The latter is a time- and resource-demanding process, which often includes multistep organic synthesis. We hypothesized that this process could be replaced by making use of liver microsomes, an in vitro system that mimics metabolism. In this study, rat liver microsomes were used to prepare radiometabolites of the dopamine transporter radioligand [18F]FE-PE2I for in vitro imaging using autoradiography and in vivo imaging using PET in rats and nonhuman primates. The primary investigated hydroxy-metabolite [18F]FE-PE2I-OH ([18F]2) was obtained in a 2% radiochemical yield and >99% radiochemical purity. In vitro and in vivo imaging demonstrated that [18F]2 readily crossed the blood-brain barrier and bound specifically and reversibly to the dopamine transporter. In conclusions, the current study demonstrates the potential of liver microsomes in the production of radiometabolites for translational imaging studies and radioligand discovery.

Correlation between insulin-degrading enzyme versus total tau and selected cytokines in patients with Alzheimer´s disease compared to non-demented controls.

OBJECTIVE: It has been increasingly recognized that the pathological progress of Alzheimer´s disease (AD) is connected to metabolic function and inflammation. Insulin-degrading enzyme (IDE) is essential for glucose metabolism and the degradation of amyloid-ß. We aimed to explore the associations between IDE, total tau, and cytokines levels in plasma from subjects with AD and non-demented controls. METHODS AND MATERIAL: Plasma samples (18 patients diagnosed with AD and 6 non-demented controls) from the Netherlands Brain Bank were used to analyze IDE levels and total tau with an enzyme-linked immunosorbent assay. Cytokines were analyzed with Luminex custom plex assays for interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α). Results were analyzed using the Mann-Whitney U and Spearman´s rank correlation tests. RESULTS: Total tau in plasma was significantly increased in AD subjects compared to non-demented control subjects (p = 0.044). Total tau was positively correlated with IDE levels in plasma in all subjects (r = 0.494, p = 0.017). Significant correlations could be demonstrated between plasma levels of IDE and IL-6 (r = 0.546, p = 0.019), IL-8 (r = 0.664, p = 0.003), IL-10 (r = 0.833, p < 0.001), and TNF-α (r = 0.633, p = 0.005) in subjects with AD, but not in non-demented controls. CONCLUSION: Results from this study suggest that plasma IDE levels may be associated with inflammation and neurodegeneration and could potentially be a target for future diagnostic and treatment strategies.

Associations between the Use of Metformin and Behavioral and Psychological Symptoms in Patients with Alzheimer´s Disease, and Type 2 Diabetes Mellitus - A Register-based Study.

BACKGROUND: Metformin, the first-line anti-diabetic drug treatment in patients with type 2 diabetes mellitus (T2DM), is suggested to be anti-inflammatory, antioxidative, and improve cognitive function, making it a promising contribution to treating Alzheimer´s disease (AD). However, the effect of metformin on behavioral and psychological symptoms of dementia (BPSD) in patients with AD has not been explored. OBJECTIVE: To investigate the associations between metformin and BPSD in patients with AD and T2DM and explore possible interaction with other antidiabetic drugs. METHODS: This cross-sectional study was based on data from the Swedish BPSD register. A total of 3745 patients with AD and antidiabetic drug treatment were included. Associations and interactions between antidiabetic drugs and BPSD were investigated by binary logistic regression. RESULTS: The use of metformin was associated with lower odds for symptoms of depression (OR 0.77, CI (95%) 0.61-0.96, p = 0.022) and anxiety (OR 0.74, CI (95%) 0.58-0.94, p = 0.015) after adjustment for age, gender, specific diagnosis, and drugs. We could not demonstrate this association with another antidiabetic drug. Interaction effects were limited to an increasing association in eating and appetite disorders using metformin and other antidiabetic drugs (i.e., drugs other than insulin, sulfonylurea, or dipeptidyl peptidase-4 inhibitors). CONCLUSION: The result of this study suggests that metformin could be beneficial for patients diagnosed with AD, other than for blood glucose control. Although, more knowledge is needed before assigning metformin a role in treating BPSD.

Distinct eicosanoid patterns in severe recalcitrant nasal polyposis.

BACKGROUND: Although altered eicosanoid levels are related to disease severity in chronic rhinosinusitis with nasal polyps (CRSwNP), identifying patients prone to recurrent nasal polyps (NPs) is still difficult. We investigated levels of nasally secreted eicosanoids before and after NP surgery in patients with or without NP recurrence (NPR) and explored potential endotypes based on pre-surgical eicosanoid levels. METHODS: Levels of leukotriene (LT) E4 , LTB4 , prostaglandin (PG) D2 , PGE2 and 15(S) hydroxyeicosatetraenoic acid (15[S]-HETE) were measured in nasal secretions with specific immunoassays at pre-surgery (n = 38) and 6 and 12 months post-surgery (n = 35), with NPR identified endoscopically. Pre- and post-surgical levels were compared between patients with and without NPR. Eicosanoid patterns among patients were explored with cluster analysis and evaluated with clinical parameters. RESULTS: Patients with recurrent NPs had pronounced pre-surgical levels of nasal 15(S)-HETE, PGD2 and LTE4 . From pre-surgery to 12 months post-surgery, NPR was associated with significant decreases of 15(S)-HETE and PGD2 relative to non-recurrence, whereas levels of LTE4 decreased at 6 months but increased again at 12 months. Clustering revealed three potential endotypes. Clusters 1 and 3 featured high and low eicosanoid levels, respectively. Cluster 2 had higher levels of LTE4 and PGD2 , lower levels of PGE2 and LTB4 , and more cases of recurrent NPs and previous NP surgeries. CONCLUSION: Elevated nasal LTE4 12 months post-surgery in NP recurrent subjects suggests that postoperative LTE4 measurements may indicate rapid NP regrowth. A distinct nasal eicosanoid profile may be used for the identification of the most severe recalcitrant patients in need of targeted immunomodulatory therapies.

Levels of eicosanoids in nasal secretions associated with nasal polyp severity in chronic rhinosinusitis.

Severe nasal polyposis and mucosal inflammation, in patients with chronic rhinosinusitis (CRS) may include a dysregulated eicosanoid profile, but a clinical role for eicosanoids in CRS with nasal polyps (NP; CRSwNP) remains to be elucidated. This study focused on assessing levels and clinical implications of inflammatory mediators in nasal secretions and urine from patients with different NP severity or Aspirin Exacerbated Respiratory Disease (AERD). Levels of leukotrienes E4 and B4, prostaglandins D2 and E2 as well as 15(S)-hydroxyeicosatetraenoic acid were measured with enzyme immunoassays and cytokines with magnetic bead immunoassays. Patients with CRSwNP were subdivided based on NP score; CRSwNP-low (NP score ≤ 4, n = 11) or CRSwNP-high (NP score ≥ 5, n = 32) and compared to CRS without polyps (CRSsNP, n = 12), CRSwNP-AERD (n = 11) and individuals without CRS (n = 25). Smell test score, fractional exhaled nitric oxide (FeNO), blood eosinophils and Sinonasal outcome test-22 were assessed as clinical markers. Leukotriene E4, prostaglandin D2 and 15(S)-hydroxyeicosatetraenoic acid in nasal secretions correlated with NP score. Nasal leukotriene E4 also correlated with FeNO and smell test score, with highest levels found in CRSwNP-AERD. Levels of prostaglandin D2 in nasal secretion as well as urinary levels of the prostaglandin D2 metabolite 11ß-prostaglandin F2α differed between CRSNP-high and CRSwNP-low. Urinary 11ß-prostaglandin F2α was associated with asthma comorbidity whereas a similar association with prostaglandin D2 in nasal secretions was not observed. In conclusion, subdividing patients based on NP severity in combination with analysis of eicosanoids in non-invasively collected nasal secretions, may have clinical implications when assessing CRS disease severity.

Increased levels of insulin-degrading enzyme in patients with type 2 diabetes mellitus.

PURPOSE: Decreasing levels of serum insulin-degrading enzyme (IDE) have been associated with an increased risk for Alzheimer´s disease (AD) in patients with type 2 diabetes mellitus (T2DM). Research on serum IDE levels in patients with T2DM is sparse and the aim of this study was to explore serum levels of IDE in patients with T2DM. METHOD: Blood serum samples were obtained from a biobank. Samples from subjects with T2DM and without metabolic disease were divided into subgroups; lifestyle treatment (n = 10), oral antidiabetic treatment (n = 17), insulin treatment (n = 20) and metabolically healthy controls (n = 18). Serum levels of IDE were analysed using specific ELISA assays. RESULTS: Serum levels of IDE were elevated in subjects with T2DM compared to metabolically healthy individuals (p = 0.033). No significant differences were detected between treatment subgroups. CONCLUSION: The present study indicates that patients with T2DM have increased serum IDE levels, compared to metabolically healthy individuals. However, for IDE to be clinically useful as a biomarker, its full function and possible use needs to be further elucidated in larger studies showing reproducible outcomes.

Serotonin 1B receptor density mapping of the human brainstem using positron emission tomography and autoradiography.

The serotonin 1B (5-HT1B) receptor has lately received considerable interest in relation to psychiatric and neurological diseases, partly due to findings based on quantification using Positron Emission Tomography (PET). Although the brainstem is an important structure in this regard, PET radioligand binding quantification in brainstem areas often shows poor reliability. This study aims to improve PET quantification of 5-HT1B receptor binding in the brainstem.Volumes of interest (VOIs) were selected based on a 3D [3H]AZ10419369 Autoradiography brainstem model, which visualized 5-HT1B receptor distribution in high resolution. Two previously developed VOI delineation methods were tested and compared to a conventional manual method. For a method based on template data, a [11C]AZ10419369 PET template was created by averaging parametric binding potential (BPND) images of 52 healthy subjects. VOIs were generated based on a predefined volume and BPND thresholding and subsequently applied to test-retest [11C]AZ10419369 parametric BPND images of 8 healthy subjects. For a method based on individual subject data, VOIs were generated directly on each individual parametric image.Both methods showed improved reliability compared to a conventional manual VOI. The VOIs created with [11C]AZ10419369 template data can be automatically applied to future PET studies measuring 5-HT1B receptor binding in the brainstem.

Hot saline irrigation in comparison to nasal packing after sinus surgery.

OBJECTIVES: Previous studies have shown that hot saline solution (HSS) nasal irrigation is effective against nasal bleeding and is used to treat nasal hemorrhage. In a pilot study, we evaluated hot saline nasal irrigation in comparison to a routinely used nasal packing in terms of self-reported complications and mucosal healing after functional endoscopic sinus surgery. METHODS: Patients undergoing surgery for bilateral chronic rhinosinusitis received polyvinyl acetate (PVA) nasal packing in the left nostril, and the right nostril was rinsed with 47°C sterile saline immediately after surgery. Patients' experiences of pain, bleeding, and other types of uncomfortable experiences were measured using a visual analog scale for each nostril before, during, and immediately after nasal packing removal. Two weeks post-surgery, the assessments were repeated including an endoscopic evaluation of the mucosa by the surgeon. RESULTS: Twenty-seven patients completed the study. Prior to removal of the packing, the patients experienced significantly more pain and other uncomfortable experiences in the nostril treated with nasal packing, as compared to the nostril solely rinsed with hot saline. After removal, patients reported significantly more uncomfortable experiences from the packing treated nostril. Two weeks post-surgery, no difference in mucosal healing was observed between the two nostrils. CONCLUSIONS: The results from this study indicate that irrigation with HSS could be an alternative postoperative treatment to conventional PVA nasal packing. Hot saline irrigation may contribute to patients experiencing improved control of postoperative bleeding, pain, and less suffering of other causes as well as health-economic benefits, without affecting the mucosal healing up to 2 weeks post-surgery. LEVEL OF EVIDENCE: 1b.

GRK3 deficiency elicits brain immune activation and psychosis.

The G protein-coupled receptor kinase (GRK) family member protein GRK3 has been linked to the pathophysiology of schizophrenia and bipolar disorder. Expression, as well as protein levels, of GRK3 are reduced in post-mortem prefrontal cortex of schizophrenia subjects. Here, we investigate functional behavior and neurotransmission related to immune activation and psychosis using mice lacking functional Grk3 and utilizing a variety of methods, including behavioral, biochemical, electrophysiological, molecular, and imaging methods. Compared to wildtype controls, the Grk3-/- mice show a number of aberrations linked to psychosis, including elevated brain levels of IL-1ß, increased turnover of kynurenic acid (KYNA), hyper-responsiveness to D-amphetamine, elevated spontaneous firing of midbrain dopamine neurons, and disruption in prepulse inhibition. Analyzing human genetic data, we observe a link between psychotic features in bipolar disorder, decreased GRK expression, and increased concentration of CSF KYNA. Taken together, our data suggest that Grk3-/- mice show face and construct validity relating to the psychosis phenotype with glial activation and would be suitable for translational studies of novel immunomodulatory agents in psychotic disorders.

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.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

In vitro phosphodiesterase 10A (PDE10A) binding in whole hemisphere human brain using the PET radioligand [18F]MNI-659.

Highly specific and sensitive biomarkers for pathologies related to dysfunctions in the basal ganglia circuit are of great value to assess therapeutic efficacy not only clinically to establish an early diagnosis, but also in terms of monitoring the efficacy of therapeutic interventions and decelerated neurodegeneration. The phosphodiesterase 10A (PDE10A) enzyme plays a central role in striatal signaling and is implicated in several neuropsychiatric disorders involving striatal pathology, such as Huntingtons disease (HD) and schizophrenia. Inhibition of PDE10A activates the neurons in the striatum and consequently leads to alteration of behavioral aspects modulated by the striatal circuit. [18F]MNI-659, (2-(2-(3-(4-(2-[18F]fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione), is a newly developed PET radioligand that shows a high binding to PDE10A in the human brain in vivo. In the present study, we examined the in vitro binding of [18F]MNI-659 in human postmortem brain to gain a better understanding of the presence, density, disease-related alterations and therapy related to changes in PDE10A expression. The results show high specific binding of [18F]MNI-659 in the caudate nucleus, putamen and the hippocampal formation. Low specific [18F]MNI-659 binding was detected in nucleus accumbens in comparison to the caudate nucleus and putamen. In vitro binding studies with [18F]MNI-659 will facilitate in elucidating better understanding of the role of PDE10A activity in health and disease that may lead to new diagnostic opportunities in HD.

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.

Discovery of a Novel Muscarinic Receptor PET Radioligand with Rapid Kinetics in the Monkey Brain.

Positron emission tomography (PET), together with a suitable radioligand, is one of the more prominent methods for measuring changes in synaptic neurotransmitter concentrations in vivo. The radioligand of choice for such measurements on the cholinergic system is the muscarinic receptor antagonist N-[1-11C]propyl-3-piperidyl benzilate (PPB). In an effort to overcome the shortcomings with the technically cumbersome synthesis of [11C]PPB, we designed and synthesized four structurally related analogues of PPB, of which (S,R)-1-methylpiperidin-3-yl)2-cyclopentyl-2-hydroxy-2-phenylacetate (1) was found to bind muscarinic receptors with similar affinity as PPB (3.5 vs 7.9 nM, respectively). (S,R)-1 was radiolabeled via N-11C-methylation at high radiochemical purity (>99%) and high specific radioactivity (>130 GBq/µmol). In vitro studies by autoradiography on human brain tissue and in vivo studies by PET in nonhuman primates demonstrated excellent signal-to-noise ratios and a kinetic profile in brain comparable to that of [11C]PBB. (S,R)-[11C]1 is a promising candidate for measuring changes in endogenous acetylcholine concentrations.

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.