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Background: Understanding patterns of association between CSF phosphorylated tau (p-tau) species and clinical disease severity will aid Alzheimer’s disease (AD) diagnosis and treatment. Objective: To evaluate changes in tau phosphorylation ratios to brain imaging (amyloid PET, [18F]GTP1 PET, and MRI) and cognition across clinical stages of AD in two different cohorts. Methods: A mass spectrometry (MS)-based method was used to evaluate the relationship between p-tau/tau phosphorylation ratios on 11 sites in CSF and AD pathology measured by tau PET ([18F]GTP1) and amyloid PET ([18F]florbetapir or [18F]florbetaben). Cohort A included cognitively normal amyloid negative (n = 6) and positive (n = 5) individuals, and amyloid positive prodromal (n = 13), mild (n = 12), and moderate AD patients (n = 10); and Cohort B included amyloid positive prodromal (n = 24) and mild (n = 40) AD patients. Results: In this cross-sectional analysis, we identified clusters of phosphosites with different profiles of phosphorylation ratios across stages of disease. Eight of 11 investigated sites were hyperphosphorylated and associated with SUVR measures from [18F]GTP1 and amyloid PET. Novel sites 111, 153, and 208 may be relevant biomarkers for AD diagnosis to complement tau hyperphosphorylation measures on previously established sites 181, 205, 217, and 231. Hypophosphorylation was detected on residues 175, 199, and 202, and was inversely associated with [18F]GTP1 and amyloid PET. Conclusion: Hyperphosphorylated and hypophosphorylated forms of tau are associated with AD pathologies, and due to their different site-specific profiles, they may be used in combination to assist with staging of disease.

ImportanceAlzheimer disease (AD), a neurodegenerative disease characterized by β-amyloid plaques and τ tangles in the brain, represents an unmet medical need with no fully approved therapeutics to modify disease progression.ObjectiveTo investigate the safety and efficacy of crenezumab, a humanized monoclonal immunoglobulin G4 antibody targeting β-amyloid oligomers, in participants with prodromal to mild (early) AD.Design, Setting, and ParticipantsTwo phase 3 multicenter randomized double-blind placebo-controlled parallel-group efficacy and safety studies of crenezumab in participants with early AD, CREAD and CREAD2, were initiated in 2016 and 2017, respectively, and were designed to evaluate the efficacy and safety of crenezumab in participants with early AD. CREAD (194 sites in 30 countries) and CREAD2 (209 sites in 27 countries) were global multicenter studies. A total of 3736 and 3664 participants were screened in CREAD and CREAD2, respectively. A total of 3736 and 3664 participants were screened in CREAD and CREAD2, respectively. Both trials enrolled individuals aged 50 to 85 years with early AD. Participants with some comorbidities and evidence of cerebral infarction or more than 4 microbleeds or areas of leptomeningeal hemosiderosis on magnetic resonance imaging were excluded. After 2923 and 2858 were excluded, respectively, 813 participants in CREAD and 806 in CREAD2 were randomly assigned in a 1:1 ratio to either placebo or crenezumab. In the final analysis, there were 409 participants in the placebo group and 404 in the crenezumab group in CREAD and 399 in the placebo group and 407 in the crenezumab group in CREAD2. Data were analyzed up until January 2019 and August 2019, respectively.InterventionsParticipants received placebo or 60 mg/kg crenezumab intravenously every 4 weeks for up to 100 weeks.Main Outcomes and MeasuresThe primary outcome was change from baseline to week 105 in Clinical Dementia Rating–Sum of Boxes (CDR-SB) score.ResultsThere were 813 participants in CREAD (mean [SD] age, 70.7 [8.2] years; 483 female and 330 male) and 806 in CREAD2 (mean [SD] age, 70.9 [7.7] years; 456 female and 350 male). Baseline characteristics were balanced between both groups. The between-group difference in mean change from baseline in CDR-SB score (placebo minus crenezumab) was −0.17 (95% CI, −0.86 to 0.53; P = .63) at week 105 in the CREAD study (88 placebo; 86 crenezumab). Compared with previous trials, no new safety signals were identified, and amyloid-related imaging abnormalities with edema were rare, mild, and transient. No meaningful changes in AD biomarkers were observed. Both studies were discontinued following a preplanned interim analysis indicating that CREAD was unlikely to meet the primary end point.Conclusions and RelevanceCrenezumab was well tolerated but did not reduce clinical decline in participants with early AD.Trial RegistrationClinicalTrials.gov Identifiers: CREAD, NCT02670083; CREAD2, NCT03114657

Purpose: ERK1/2 signaling can be dysregulated in cancer. GDC-0994 is an oral inhibitor of ERK1/2. A first-in-human, phase I dose escalation study of GDC-0994 was conducted in patients with locally advanced or metastatic solid tumors. Patients and Methods: GDC-0994 was administered once daily on a 21-day on/7-day off schedule to evaluate safety, pharmacokinetics, and preliminary signs of efficacy. Patients with pancreatic adenocarcinoma and BRAF-mutant colorectal cancer were enrolled in the expansion stage. Results: Forty-seven patients were enrolled in six successive cohorts (50–800 mg). A single DLT of grade 3 rash occurred at 600 mg. The most common drug-related adverse events (AE) were diarrhea, rash, nausea, fatigue, and vomiting. Pharmacokinetic data showed dose-proportional increases in exposure, with a mean half-life of 23 hours, supportive of once daily dosing. In evaluable paired biopsies, MAPK pathway inhibition ranged from 19% to 51%. Partial metabolic responses by FDG-PET were observed in 11 of 20 patients across dose levels in multiple tumor types. Overall, 15 of 45 (33%) patients had a best overall response of stable disease and 2 patients with BRAF-mutant colorectal cancer had a confirmed partial response. Conclusions: GDC-0994 had an acceptable safety profile and pharmacodynamic effects were observed by FDG-PET and in serial tumor biopsies. Single-agent activity was observed in 2 patients with BRAF-mutant colorectal cancer.

The histamine 3 (H3) receptor is a presynaptic autoreceptor in the central nervous system that regulates the synthesis and release of histamine and modulates the release of other major neurotransmitters. H3 receptor inverse agonists (IAs) may be efficacious in the treatment of various central nervous system disorders, including excessive daytime sleepiness, attention deficit hyperactivity disorder, Alzheimer disease, ethanol addiction, and obesity. Methods: Using PET and a novel high-affinity and selective radioligand 11C-MK-8278, we studied the tracer biodistribution, quantification, and brain H3 receptor occupancy (RO) of MK-0249 and MK-3134, 2 potential IA drugs targeting cerebral H3 receptors, in 6 healthy male subjects (age, 19–40 y). The relationship among H3 IA dose, time on target, and peripheral pharmacokinetics was further investigated in 15 healthy male volunteers (age, 18–40 y) with up to 3 PET scans and 3 subjects per dose level. Results: The mean effective dose for 11C-MK-8278 was 5.4 ± 1.1 μSv/MBq. Human brain kinetics showed rapid high uptake and fast washout. Binding potential values can be assessed using the pons as a reference region, with a test–retest repeatability of 7%. Drug RO data showed low interindividual variability per dose (mean RO SD, 2.1%), and a targeted 90% RO can be reached for both IAs at clinically feasible doses. Conclusion:11C-MK-8278 is a useful novel PET radioligand for determination of human cerebral H3 receptor binding and allows highly reproducible in vivo brain occupancy of H3-targeting drugs, hereby enabling the evaluation of novel compounds in early development to select doses and schedules.

Reduced NMDA receptor functioning is hypothesized to underlie the cognitive and negative symptoms associated with schizophrenia. However, because direct activation of the NMDA receptor is accompanied by neurotoxicity, mechanisms that activate the glycine co-agonist site on the NMDA receptor could carry greater therapeutic potential. In the current study, the effects of two glycine transporter 1 (GlyT1) inhibitors, RG1678 and ORG25935, were characterized in the object-retrieval detour (ORD) task in scopolamine-impaired rhesus monkeys and, using positron emission tomography (PET), the GlyT1 occupancy to efficacy relationship of each compound was established. Scopolamine exerted a significant decrease in accuracy in the ORD task. Lower doses of RG1678 (0.3 and 1.0 mg/kg, p.o.) significantly attenuated the impact of scopolamine, whereas the highest dose tested (1.8 mg/kg) did not. The predicted GlyT1 occupancies of RG1678 at the effective doses were ~10 and 30 %. ORG25935 (0.1, 0.3, and 1 mg/kg, p.o.) also significantly attenuated the impact of scopolamine on the ORD task, whereas 3 mg/kg did not. The predicted GlyT1 occupancies of ORG25935 at the effective doses ranged from 16 to 80 %. These data suggest that GlyT1 inhibitors have the potential to improve performance on prefrontal cortex-dependent tests such as the ORD task, but that efficacy is lost when higher occupancies are achieved. Importantly, recent Ph2B data published by Roche suggests that low but not high doses of RG1678 improved negative symptoms in patients with schizophrenia, highlighting the potential translational nature of the current preclinical findings.

In the accompanying paper we describe how MRK-409 unexpectedly produced sedation in man at relatively low levels of GABAA receptor occupancy (∼10%). Since it was not clear whether this sedation was mediated via the α2/α3 or α1 GABAA subtype(s), we characterized the properties of TPA023B, a high-affinity imidazotriazine which, like MRK-409, has partial agonist efficacy at the α2 and α3 subtype but is an antagonist at the α1 subtype, at which MRK-409 has weak partial agonism. TPA023B gave dose- and time-dependent occupancy of rat brain GABAA receptors as measured using an in vivo [3H]flumazenil binding assay, with 50% occupancy corresponding to a respective dose and plasma drug concentration of 0.09 mg/kg and 19 ng/mL, the latter of which was similar to that observed in mice (25 ng/mL) and comparable to values obtained in baboon and man using [11C]flumazenil PET (10 and 5.8 ng/mL, respectively). TPA023B was anxiolytic in rodent and primate (squirrel monkey) models of anxiety (elevated plus maze, fear-potentiated startle, conditioned suppression of drinking, conditioned emotional response) yet had no significant effects in rodent or primate assays of ataxia and/or myorelaxation (rotarod, chain-pulling, lever pressing), up to doses (10 mg/kg) corresponding to occupancy of greater than 99%. In man, TPA023B was well tolerated at a dose (1.5 mg) that produced occupancy of >50%, suggesting that the sedation previously seen with MRK-409 is due to the partial agonist efficacy of that compound at the α1 subtype, and highlighting the importance of antagonist efficacy at this particular GABAA receptor population for avoiding sedation in man.

MRK-409 binds to α1-, α2-, α3- and α5-containing human recombinant GABAA receptors with comparable high affinity (0.21–0.40 nM). However, MRK-409 has greater agonist efficacy at the α3 compared with α1 subtypes (respective efficacies relative to the full agonist chlordiazepoxide of 0.45 and 0.18). This compound readily penetrates the brain in rats and occupies the benzodiazepine site of GABAA receptors, measured using an in vivo [3H]flumazenil binding assay, with an Occ50 of 2.2 mg/kg p.o. and a corresponding plasma EC50 of 115 ng/mL. Behaviourally, the α3-preferring agonist efficacy profile of MRK-409 produced anxiolytic-like activity in rodent and primate unconditioned and conditioned models of anxiety with minimum effective doses corresponding to occupancies, depending on the particular model, ranging from ∼35% to 65% yet there were minimal overt signs of sedation at occupancies greater than 90%. In humans, however, safety and tolerability studies showed that there was pronounced sedation at a dose of 2 mg, resulting in a maximal tolerated dose of 1 mg. This 2 mg dose corresponded to a Cmax plasma concentration of 28 ng/mL, which, based on the rodent plasma EC50 for occupancy of 115 ng/mL, suggested that sedation in humans occurs at low levels of occupancy. This was confirmed in human positron emission tomography studies, in which [11C]flumazenil uptake following a single dose of 1 mg MRK-409 was comparable to that of placebo, indicating that occupancy of GABAA receptor benzodiazepine binding sites by MRK-409 was below the limits of detection (i.e.

Fluorine-18 fluoroethylflumazenil ([18F]FEF) is a tracer for central benzodiazepine (BZ) receptors which is proposed as an alternative to carbon-11 flumazenil for in vivo imaging using positron emission tomography (PET) in humans. In this study, [18F]FEF kinetic data were acquired using a 60-min two-injection protocol on three normal subjects and two patients suffering from mesiotemporal epilepsy as demonstrated by abnormal magnetic resonance imaging and [18F]fluorodeoxyglucose positron emission tomography. First, a tracer bolus injection was performed and [18F]FEF rapidly distributed in the brain according to the known BZ receptor distribution. Thirty minutes later a displacement injection of 0.01 mg/kg of unlabelled flumazenil was performed. Activity was rapidly displaced from all BZ receptor regions demonstrating the specific binding of [18F]FEF. No displacement was observed in the pons. Plasma input function was obtained from arterial blood sampling, and metabolite analysis was performed by high-performance liquid chromatography. Metabolite quantification revealed a fast decrease in tracer plasma concentration, such that at 5 min post injection about 70% of the total radioactivity in plasma corresponded to [18F]FEF, reaching 24% at 30 min post injection. The interactions between [18F]FEF and BZ receptors were described using linear compartmental models with plasma input and reference tissue approaches. Binding potential values were in agreement with the known distribution of BZ receptors in human brain. Finally, in two patients with mesiotemporal sclerosis, reduced uptake of [18F]FEF was clearly observed in the implicated left hippocampus.

In this work, a mathematical correction for metabolites has been validated which estimates the relative amount of [11C]flumazenil ([11C]FMZ) in the total plasma curve from the tissue kinetic data without the need for direct metabolite measurement in blood plasma samples. Kinetic data were obtained using a 90-min three-injection protocol on five normal volunteers. First, the relative amount of [11C]FMZ in plasma was modelled by a two-parameter exponential function. The parameters were estimated either directly by fitting this model to the blood plasma metabolite measurements, or indirectly from the simultaneous fitting of tissue time activity curves from several brain regions with a non-linear FMZ kinetic model. Second, the direct and indirect metabolite corrections were fixed and the FMZ compartmental parameters were determined on a regional basis in the brain. The validation was performed by comparing the regional values of benzodiazepine receptor density Bmax and equilibrium dissociation constant Kd obtained with the direct metabolite correction with those values obtained with the indirect correction. For Bmax, the correlation coefficient r2 was above 0.97 for all subjects and the slope values of the linear regression were within the interval [0.97, 1.2]. For Kd, r2 was above 0.96, and the slope values of the linear regression were within the interval [0.99, 1.1]. Simulation studies were performed in order to evaluate whether this metabolite correction method could be used in a clinical protocol where only a single [11C]FMZ injection and a linear compartmental model are used. The resulting [11C]FMZ distribution volume estimates were found to be linearly correlated with the true values, with r2=1.0 and a slope value of 1.1. The mathematical metabolite correction proved to be a feasible and reliable method to estimate the relative amount of [11C]FMZ in plasma and the compartmental model parameters for three-injection protocols. Although validation with real data is necessary, simulation results suggest that our analysis method may also be applied to single-injection protocols.

A novel, high-affinity (.7-2.0 nmol) compound that selectively activates the alpha2, alpha 3, and alpha 5 (but not alpha1) gamma-aminobutyric acid-A (GABA(A)) receptor subtypes, TPA023B (2',6-difluoro-5'-[3-(1-hydroxy-1-methylethyl) imidazo[1,2-b][1,2,4]triazin-7-yl][1,1'-biphenyl]-2-carbonitrile) was pharmacologically characterized and studied by means of positron emission tomography (PET) to determine dynamic occupancies of the benzodiazepine binding site of human brain GABA(A) receptors after a single oral dose.Four healthy male volunteers were studied in a double-blind, randomized placebo-controlled study of which three were given a single dose of 1.5 mg TPA023B and the fourth received placebo. The time course of GABA(A) receptor occupancy was determined with multiple dynamic [(11)C]flumazenil PET studies at pre-dose baseline and 5 and 24 hours after dose. Arterial sampling and full kinetic modeling with a two-compartment model was used to calculate parametric maps of receptor availability (distribution volume V(T)) and of occupancy.The GABA(A) receptor occupancy as determined from [(11)C]flumazenil V(T) values in all brain regions was reduced homogeneously, on average by 52.5 +/- 1.2% after 5 hours and 46.4 +/- 6.0% after 24 hours. No serious adverse events were encountered in humans.Single oral doses of 1.5 mg of TPA023B correspond to average receptor occupancies in neocortical regions of 52% and 46% after 5 and 24 hours, respectively. Provided suitable ligands and quantification methods are available for the appropriate target, quantitative PET offers a unique tool for dynamic in vivo measurement of relevant on-site receptor occupancy.

[ 18 F]MK-9470 is a selective, high-affinity, inverse agonist (human IC 50 , 0.7 nM) for the cannabinoid CB1 receptor (CB1R) that has been developed for use in human brain imaging. Autoradiographic studies in rhesus monkey brain showed that [ 18 F]MK-9470 binding is aligned with the reported distribution of CB1 receptors with high specific binding in the cerebral cortex, cerebellum, caudate/putamen, globus pallidus, substantia nigra, and hippocampus. Positron emission tomography (PET) imaging studies in rhesus monkeys showed high brain uptake and a distribution pattern generally consistent with that seen in the autoradiographic studies. Uptake was blocked by pretreatment with a potent CB1 inverse agonist, MK-0364. The ratio of total to nonspecific binding in putamen was 4–5:1, indicative of a strong specific signal that was confirmed to be reversible via displacement studies with MK-0364. Baseline PET imaging studies in human research subject demonstrated behavior of [ 18 F]MK-9470 very similar to that seen in monkeys, with very good test–retest variability (7%). Proof of concept studies in healthy young male human subjects showed that MK-0364, given orally, produced a dose-related reduction in [ 18 F]MK-9470 binding reflecting CB1R receptor occupancy by the drug. Thus, [ 18 F]MK-9470 has the potential to be a valuable, noninvasive research tool for the in vivo study of CB1R biology and pharmacology in a variety of neuropsychiatric disorders in humans. In addition, it allows demonstration of target engagement and noninvasive dose-occupancy studies to aid in dose selection for clinical trials of CB1R inverse agonists.

Serotonin-1A (5-HT(1A)) receptors are implicated in neurochemical mechanisms underlying anxiety and depression and their treatment. Animal studies have suggested that 4-(2'-methoxyphenyl)-1-[2'-[ N-(2"-pyridinyl)- p-[(18)F]fluorobenzamido] ethyl] piperazine ( p-MPPF) may be a suitable positron emission tomography (PET) tracer of 5-HT(1A) receptors. To test p-MPPF in humans, we performed 60-min dynamic PET scans in 13 healthy volunteers after single bolus injection. Metabolite quantification revealed a fast decrease in tracer plasma concentration, such that at 5 min post injection about 25% of the total radioactivity in plasma corresponded to p-MPPF. Radioactivity concentration was highest in hippocampus, intermediate in neocortex and lowest in basal ganglia and cerebellum. The interactions between p-MPPF and 5-HT(1A) receptors were described using linear compartmental models with plasma input and reference tissue approaches. The two quantification methods provided similar results which are in agreement with previous reports on 5-HT(1A) receptor brain distribution. In conclusion, our results show that p-MPPF is a suitable PET radioligand for 5-HT(1A) receptor human studies.

As a first approach to study the effect of early visual deprivation in the GABA-ergic inhibitory system, the distribution of benzodiazepine receptors (BZR) was accurately estimated using [11C]flumazenil ([11C]FMZ). Measurements were carried out in five subjects who became blind early in life and in five sighted control subjects. The interactions between [11C]FMZ and BZR were described using a non-linear compartmental analysis which permitted to estimate the BZR synaptic density independently of other model parameters. The distribution of BZR in the visual areas and other cortical regions of blind subjects was qualitatively and quantitatively similar to that of controls. However, the BZR density in the cerebellum was significantly lower in blind than in control subjects (P