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Targeting tauopathy with radioligands: Positron emission tomography (PET) can be useful for imaging the brain to study neurodegeneration but requires radioligands to identify and quantify specific proteins. Lu and colleagues developed two radioligands that detect an enzyme involved in posttranslational modifications of tau, a protein that accumulates in tangles in the brain during Alzheimer's disease. The ligands imaged and quantified the target enzyme in rodent and monkey brain tissue and postmortem brain tissue from humans with Alzheimer's disease. When delivered to healthy monkeys and humans, the radioligands showed uptake in the brain by PET imaging. These radioligands could be useful for drug development for tauopathies. We aimed to develop effective radioligands for quantifying brain O-linked-β-N-acetyl-glucosamine (O-GlcNAc) hydrolase (OGA) using positron emission tomography in living subjects as tools for evaluating drug target engagement. Posttranslational modifications of tau, a biomarker of Alzheimer's disease, by O-GlcNAc through the enzyme pair OGA and O-GlcNAc transferase (OGT) are inversely related to the amounts of its insoluble hyperphosphorylated form. Increase in tau O-GlcNAcylation by OGA inhibition is believed to reduce tau aggregation. LSN3316612, a highly selective and potent OGA ligand [half-maximal inhibitory concentration (IC50) = 1.9 nM], emerged as a lead ligand after in silico analysis and in vitro evaluations. [3H]LSN3316612 imaged and quantified OGA in postmortem brains of rat, monkey, and human. The presence of fluorine and carbonyl functionality in LSN3316612 enabled labeling with positron-emitting fluorine-18 or carbon-11. Both [18F]LSN3316612 and [11C]LSN3316612 bound reversibly to OGA in vivo, and such binding was blocked by pharmacological doses of thiamet G, an OGA inhibitor of different chemotype, in monkeys. [18F]LSN3316612 entered healthy human brain avidly (~4 SUV) without radiodefluorination or adverse effect from other radiometabolites, as evidenced by stable brain total volume of distribution (VT) values by 110 min of scanning. Overall, [18F]LSN3316612 is preferred over [11C]LSN3316612 for future human studies, whereas either may be an effective positron emission tomography radioligand for quantifying brain OGA in rodent and monkey. [ABSTRACT FROM AUTHOR]

Background and Purpose: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease. Experimental Approach We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat–human) benchmarking of structure–activity relationship molecules to clinical comparators. Key Results: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti‐targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose‐dependently occupies rodent cortical M1 receptors. Conclusions and Implications: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease. [ABSTRACT FROM AUTHOR]

Nociceptin/Orphanin FQ (N/ OFQ) is a 17 amino acid peptide whose receptor is designated ORL1 or nociceptin receptor ( NOP). We utilized a potent, selective, and orally bioavailable antagonist with documented engagement with NOP receptors in vivo to assess antidepressant- and anxiolytic-related pharmacological effects of NOP receptor blockade along with measures of cognitive and motor impingement. LY2940094 ([2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4′-piperidine]-1′-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol) displayed antidepressant-like behavioral effects in the forced-swim test in mice, an effect absent in NOP−/− mice. LY2940094 also augmented the behavioral effect of fluoxetine without changing target occupancies ( NOP and serotonin reuptake transporter [ SERT]). LY2940094 did not have effects under a differential-reinforcement of low rate schedule. Although anxiolytic-like effects were not observed in some animal models (conditioned suppression, 4-plate test, novelty-suppressed feeding), LY2940094 had effects like that of anxiolytic drugs in three assays: fear-conditioned freezing in mice, stress-induced increases in cerebellar cGMP in mice, and stress-induced hyperthermia in rats. These are the first reports of anxiolytic-like activity with a systemically viable NOP receptor antagonist. LY2940094 did not disrupt performance in either a 5-choice serial reaction time or delayed matching-to-position assay. LY2940094 was also not an activator or suppressor of locomotion in rodents nor did it induce failures of rotarod performance. These data suggest that LY2940094 has unique antidepressant- and anxiolytic-related pharmacological effects in rodents. Clinical proof of concept data on this molecule in depressed patients have been reported elsewhere. [ABSTRACT FROM AUTHOR]

Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging., Chemistry and Chemical Biology

Background and Purpose: We aimed to identify and develop novel, selective muscarinic M1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease.Experimental Approach: We developed and utilized a novel M1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators.Key Results: Using this paradigm, we identified a series of M1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M1 receptors over native M2 and M3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M1 receptors.Conclusions and Implications: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M1 receptors in physiology and disease. [ABSTRACT FROM AUTHOR]

Abstract: [11C]NOP-1A is a novel high-affinity PET ligand for imaging nociceptin/orphanin FQ peptide (NOP) receptors. Here, we report reproducibility and reliability measures of binding parameter estimates for [11C]NOP-1A binding in the brain of healthy humans. After intravenous injection of [11C]NOP-1A, PET scans were conducted twice on eleven healthy volunteers on the same (10/11 subjects) or different (1/11 subjects) days. Subjects underwent serial sampling of radial arterial blood to measure parent radioligand concentrations. Distribution volume (V T; a measure of receptor density) was determined by compartmental (one- and two-tissue) modeling in large regions and by simpler regression methods (graphical Logan and bilinear MA1) in both large regions and voxel data. Retest variability and intraclass correlation coefficient (ICC) of V T were determined as measures of reproducibility and reliability respectively. Regional [11C]NOP-1A uptake in the brain was high, with a peak radioactivity concentration of 4–7 SUV (standardized uptake value) and a rank order of putamen>cingulate cortex>cerebellum. Brain time–activity curves fitted well in 10 of 11 subjects by unconstrained two-tissue compartmental model. The retest variability of V T was moderately good across brain regions except cerebellum, and was similar across different modeling methods, averaging 12% for large regions and 14% for voxel-based methods. The retest reliability of V T was also moderately good in most brain regions, except thalamus and cerebellum, and was similar across different modeling methods averaging 0.46 for large regions and 0.48 for voxels having gray matter probability >20%. The lowest retest variability and highest retest reliability of V T were achieved by compartmental modeling for large regions, and by the parametric Logan method for voxel-based methods. Moderately good reproducibility and reliability measures of V T for [11C]NOP-1A make it a useful PET ligand for comparing NOP receptor binding between different subject groups or under different conditions in the same subject. [Copyright &y& Elsevier]

Background and Purpose: We aimed to identify and develop novel, selective muscarinic M 1 receptor agonists as potential therapeutic agents for the symptomatic treatment of Alzheimer's disease., Experimental Approach: We developed and utilized a novel M 1 receptor occupancy assay to drive a structure activity relationship in a relevant brain region while simultaneously tracking drug levels in plasma and brain to optimize for central penetration. Functional activity was tracked in relevant native in vitro assays allowing translational (rat-human) benchmarking of structure-activity relationship molecules to clinical comparators., Key Results: Using this paradigm, we identified a series of M 1 receptor selective molecules displaying desirable in vitro and in vivo properties and optimized key features, such as central penetration while maintaining selectivity and a partial agonist profile. From these compounds, we selected spiropiperidine 1 (SPP1). In vitro, SPP1 is a potent, partial agonist of cortical and hippocampal M 1 receptors with activity conserved across species. SPP1 displays high functional selectivity for M 1 receptors over native M 2 and M 3 receptor anti-targets and over a panel of other targets. Assessment of central target engagement by receptor occupancy reveals SPP1 significantly and dose-dependently occupies rodent cortical M 1 receptors., Conclusions and Implications: We report the discovery of SPP1, a novel, functionally selective, brain penetrant partial orthosteric agonist at M 1 receptors, identified by a novel receptor occupancy assay. SPP1 is amenable to in vitro and in vivo study and provides a valuable research tool to further probe the role of M 1 receptors in physiology and disease., (© 2018 The British Pharmacological Society.)

The translocator protein (TSPO) is a commonly used imaging target to investigate neuroinflammation. Although TSPO imaging demonstrates great promise, its signal exhibits substantial interindividual variability, which needs to be accounted for to uncover group effects that are truly reflective of neuroimmune activation. Recent evidence suggests that relative metrics computed using pseudoreference approaches can minimize within-group variability and increase sensitivity to detect physiologically meaningful group differences. Here, we evaluated various ratio approaches for TSPO imaging and compared them with standard kinetic modeling techniques, analyzing 2 different disease cohorts. Patients with chronic low back pain (cLBP) or amyotrophic lateral sclerosis (ALS) and matching healthy controls received 11 C-PBR28 PET scans. The occipital cortex, cerebellum and whole brain were first evaluated as candidate pseudoreference regions by testing for the absence of group differences in SUV and distribution volume ( V T ) estimated with an arterial input function. The SUV from target regions (cLBP study, thalamus; ALS study, precentral gyrus) was normalized with the SUV from candidate pseudoreference regions (i.e., occipital cortex, cerebellum, and whole brain) to obtain SUVR occip , SUVR cereb , and SUVR WB The sensitivity to detect group differences in target regions was compared using various SUVR approaches, as well as distribution volume ratio (DVR) estimated with (blDVR) or without arterial input function (refDVR), and V T Additional voxelwise SUVR group analyses were performed. We observed no significant group differences in pseudoreference V T or SUV, excepting whole-brain V T , which was higher in cLBP patients than controls. Target V T elevations in patients ( P = 0.028 and 0.051 in cLBP and ALS, respectively) were similarly detected by SUVR occip and SUVR WB , and by refDVR and blDVR (less reliably by SUVR cereb ). In voxelwise analyses, SUVR occip , but not SUVR cereb , identified regional group differences initially observed with SUVR WB , and in additional areas suspected to be affected in the pathology examined. All ratio metrics were highly cross-correlated, but generally were not associated with V T. Although important caveats need to be considered when using relative metrics, ratio analyses appear to be similarly sensitive to detect pathology-related group differences in 11 C-PBR28 signal as classic kinetic modeling techniques. The occipital cortex may be a suitable pseudoreference region, at least for the populations evaluated, pending further validation in larger cohorts., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Hydroxamic acid-based histone deacetylase inhibitors (HDACis) are a class of molecules with therapeutic potential currently reflected in the use of suberoylanilide hydroxamic acid (SAHA; Vorinostat) to treat cutaneous T-cell lymphomas (CTCL). HDACis may have utility beyond cancer therapy, as preclinical studies have ascribed HDAC inhibition as beneficial in areas such as heart disease, diabetes, depression, neurodegeneration, and other disorders of the central nervous system (CNS). However, little is known about the pharmacokinetics (PK) of hydroxamates, particularly with respect to CNS-penetration, distribution, and retention. To explore the rodent and non-human primate (NHP) brain permeability of hydroxamic acid-based HDAC inhibitors using positron emission tomography (PET), we modified the structures of belinostat (PXD101) and panobinostat (LBH-589) to incorporate carbon-11. We also labeled PCI 34051 through carbon isotope substitution. After characterizing the in vitro affinity and efficacy of these compounds across nine recombinant HDAC isoforms spanning Class I and Class II family members, we determined the brain uptake of each inhibitor. Each labeled compound has low uptake in brain tissue when administered intravenously to rodents and NHPs. In rodent studies, we observed that brain accumulation of the radiotracers were unaffected by the pre-administration of unlabeled inhibitors. Knowing that CNS-penetration may be desirable for both imaging applications and therapy, we explored whether a liquid chromatography, tandem mass spectrometry (LC-MS-MS) method to predict brain penetrance would be an appropriate method to pre-screen compounds (hydroxamic acid-based HDACi) prior to PET radiolabeling. LC-MS-MS data were indeed useful in identifying additional lead molecules to explore as PET imaging agents to visualize HDAC enzymes in vivo. However, HDACi brain penetrance predicted by LC-MS-MS did not strongly correlate with PET imaging results. This underscores the importance of in vivo PET imaging tools in characterizing putative CNS drug lead compounds and the continued need to discover effect PET tracers for neuroepigenetic imaging.

Some recently published in vitro studies with two metabotropic glutamate 2/3 receptor (mGluR(2/3)) agonists [(-)-2-oxa-4-aminobicyclo[3.1.0] hexane-4,6-dicarboxylic acid (LY379268) and 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate (LY354740)] suggest that these compounds may also directly interact with dopamine (DA) D(2) receptors. The current in vitro and in vivo studies were undertaken to further explore this potential interaction with D(2) receptors. LY379268 and LY354740 failed to inhibit D(2) binding in both native striatal tissue homogenates and cloned receptors at concentrations up to 10 microM. LY379268 and LY354740 (up to 10 microM) also failed to stimulate [(35)S]GTPgammaS binding in D(2L)- and D(2S)-expressing clones in the presence of NaCl or N-methyl-d-glucamine. In an in vivo striatal D(2) receptor occupancy assay, LY379268 (3-30 mg/kg) or LY354740 (1-10 mg/kg) failed to displace raclopride (3 microg/kg i.v.), whereas aripiprazole (10-60 mg/kg) showed up to 90% striatal D(2) receptor occupancy. LY379268 (10 mg/kg) and raclopride (3 mg/kg) blocked d-amphetamine and phencyclidine (PCP)-induced hyperactivity in wild-type mice. However, the effects of LY379268 were lost in mGlu(2/3) receptor knockout mice. In DA D(2) receptor-deficient mice, LY379268 but not raclopride blocked both PCP and d-amphetamine-evoked hyperactivity. In the striatum and nucleus accumbens, LY379268 (3 and 10 mg/kg) was without effect on the DA synthesis rate in reserpinized rats and also failed to prevent S-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine-induced reductions in DA synthesis rate. Taken together, the current data fail to show evidence of direct DA D(2) receptor interactions of LY379268 and LY354740 in vitro or in vivo. Instead, these results provide further evidence for a novel antipsychotic mechanism of action for mGluR(2/3) agonists.