Design, Synthesis, and Evaluation of Hydroxamic Acid-Based Molecular Probes for In Vivo Imaging of Histone Deacetylase (HDAC) in Brain

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 Biolog

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer’s disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including ‘epileptic-like’ seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD

Original Article Design, synthesis, and evaluation of hydroxamic acid-based molecular probes for in vivo imaging of histone deacetylase (HDAC) in brain

Abstract: 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

Pseudo-reference regions for glial imaging with (11)C-PBR28:investigation in two clinical cohorts

none14The translocator protein (TSPO) is a commonly used imaging target to investigate neuroinflammation. While 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 pseudo-reference 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 two 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. Occipital cortex, cerebellum and whole brain were first evaluated as candidate pseudo-reference regions by testing for the absence of group differences in Standardized Uptake Value (SUV) and distribution volume (VT) estimated with an arterial input function (AIF). SUV from target regions (cLBP study - thalamus; ALS study - precentral gyrus) was normalized with SUV from candidate pseudo-reference regions to obtain SUVRoccip, SUVRcereb, and SUVRWB 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 AIF (refDVR), and VT Additional voxelwise SUVR group analyses were performed. We observed no significant group differences in pseudo-reference VT or SUV, excepting whole-brain VT, which was higher in cLBP patients than controls. Target VT elevations in patients (P = 0.028 and 0.051 in cLBP and ALS, respectively) were similarly detected by SUVRoccip and SUVRWB, and by refDVR and blDVR (less reliably by SUVRcereb). In voxelwise analyses, SUVRoccip, but not SUVRcereb, identified regional group differences initially observed with SUVRWB, 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 VT While 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. Occipital cortex may be a suitable pseudo-reference region, at least for the populations evaluated, pending further validation in larger cohorts.noneAlbrecht, Daniel Strakis; Normandin, Marc David; Shcherbinin, Sergey; Wooten, Dustin W; Schwarz, Adam J; Zurcher, Nicole R; Barth, Vanessa N; Guehl, Nicolas J; Johnson-Akeju, Oluwaseun; Atassi, Nazem; Veronese, Mattia; Turkheimer, Federico; Hooker, Jacob M; Loggia, Marco LucianoAlbrecht, Daniel Strakis; Normandin, Marc David; Shcherbinin, Sergey; Wooten, Dustin W; Schwarz, Adam J; Zurcher, Nicole R; Barth, Vanessa N; Guehl, Nicolas J; Johnson-Akeju, Oluwaseun; Atassi, Nazem; Veronese, Mattia; Turkheimer, Federico; Hooker, Jacob M; Loggia, Marco Lucian

Pseudoreference Regions for Glial Imaging with 11

The translocator protein (TSPO) is a commonly used imaging target to investigate neuroinflammation. While 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 pseudo-reference 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 two 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. Occipital cortex, cerebellum and whole brain were first evaluated as candidate pseudo-reference regions by testing for the absence of group differences in Standardized Uptake Value (SUV) and distribution volume (VT) estimated with an arterial input function (AIF). SUV from target regions (cLBP study - thalamus; ALS study - precentral gyrus) was normalized with SUV from candidate pseudo-reference regions to obtain SUVRoccip, SUVRcereb, and SUVRWB 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 AIF (refDVR), and VT Additional voxelwise SUVR group analyses were performed. We observed no significant group differences in pseudo-reference VT or SUV, excepting whole-brain VT, which was higher in cLBP patients than controls. Target VT elevations in patients (P = 0.028 and 0.051 in cLBP and ALS, respectively) were similarly detected by SUVRoccip and SUVRWB, and by refDVR and blDVR (less reliably by SUVRcereb). In voxelwise analyses, SUVRoccip, but not SUVRcereb, identified regional group differences initially observed with SUVRWB, 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 VT While 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. Occipital cortex may be a suitable pseudo-reference region, at least for the populations evaluated, pending further validation in larger cohorts