Treatments for cognitive deficits associated with central nervous system (CNS) disorders such as Alzheimer disease and schizophrenia remain significant unmet medical needs that incur substantial pressure on the health care system. The α7 nicotinic acetylcholine receptor (nAChR) has garnered substantial attention as a target for cognitive deficits based on receptor localization, robust preclinical effects, genetics implicating its involvement in cognitive disorders, and encouraging, albeit mixed, clinical data with α7 nAChR orthosteric agonists. Importantly, previous orthosteric agonists at this receptor suffered from off-target activity, receptor desensitization, and an inverted U-shaped dose-effect curve in preclinical assays that limit their clinical utility. To overcome the challenges with orthosteric agonists, we have identified a novel selective α7 positive allosteric modulator (PAM), BNC375. This compound is selective over related receptors and potentiates acetylcholine-evoked α7 currents with only marginal effect on the receptor desensitization kinetics. In addition, BNC375 enhances long-term potentiation of electrically evoked synaptic responses in rat hippocampal slices and in vivo. Systemic administration of BNC375 reverses scopolamine-induced cognitive deficits in rat novel object recognition and rhesus monkey object retrieval detour (ORD) task over a wide range of exposures, showing no evidence of an inverted U-shaped dose-effect curve. The compound also improves performance in the ORD task in aged African green monkeys. Moreover, ex vivo 13C-NMR analysis indicates that BNC375 treatment can enhance neurotransmitter release in rat medial prefrontal cortex. These findings suggest that α7 nAChR PAMs have multiple advantages over orthosteric α7 nAChR agonists for the treatment of cognitive dysfunction associated with CNS diseases. SIGNIFICANCE STATEMENT: BNC375 is a novel and selective α7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PAM) that potentiates acetylcholine-evoked α7 currents in in vitro assays with little to no effect on the desensitization kinetics. In vivo, BNC375 demonstrated robust procognitive effects in multiple preclinical models across a wide exposure range. These results suggest that α7 nAChR PAMs have therapeutic potential in central nervous system diseases with cognitive impairments.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

Inhibition of O-GlcNAcase (OGA) has emerged as a promising therapeutic approach to treat tau pathology in neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Beginning with carbohydrate-based lead molecules, we pursued an optimization strategy of reducing polar surface area to align the desired drug-like properties of potency, selectivity, high central nervous system (CNS) exposure, metabolic stability, favorable pharmacokinetics, and robust in vivo pharmacodynamic response. Herein, we describe the medicinal chemistry and pharmacological studies that led to the identification of (3aR,5S,6S,7R,7aR)-5-(difluoromethyl)-2-(ethylamino)-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]thiazole-6,7-diol 42 (MK-8719), a highly potent and selective OGA inhibitor with excellent CNS penetration that has been advanced to first-in-human phase I clinical trials.

Alzheimer’s disease (AD) is a chronic neurodegenerative disease with pathological hallmarks including the formation of extracellular aggregates of amyloid-beta (Aβ) known as plaques and intracellular tau tangles. Coincident with the formation of Aβ plaques is recruitment and activation of glial cells to the plaque forming a plaque niche. In addition to histological data showing the formation of the niche, AD genetic studies have added to the growing appreciation of how dysfunctional glia pathways drive neuropathology, with emphasis on microglia pathways. Genomic approaches enable comparisons of human disease profiles between different mouse models informing on their utility to evaluate secondary changes to triggers such as Aβ deposition.

MK-8719 is a selective inhibitor of the O-GlcNAcase (OGA) enzyme that is currently in Phase 1 clinical trials for the treatment of Progressive Supranuclear Palsy (PSP). Here we summarize the preclinical validation data for MK-8719 generated in Tg4510 transgenic mice as well as human tolerability and target engagement (PET) data for MK-8719 following single dose administration. Evaluation of the pharmacodynamic activity and efficacy of MK-8719 was conducted in Tg4510 transgenic mice that overexpress human tau with P301L mutation. The effects of MK-8719 on total protein O-GlcNAcylation and the accumulation of pathological species of tau were determined by immunoassay. The influence of MK-8719 on brain atrophy of Tg4510 mice was evaluated by volumetric MRI and the formation of neurofibrillary tangles was assessed using immunohistochemistry. Safety and tolerability of single doses of MK-8719 between 5 and 1200 mg were evaluated in healthy volunteers (n=16) using an alternating-panel single ascending dose design. MK-8719 target engagement in the brain was evaluated in a separate Phase 1 study using PET with [18F]MK-8553, a radiolabeled tracer of a novel small molecule inhibitor of the OGA enzyme. In vitro studies demonstrated that MK-8719 is a competitive reversible inhibitor of the human OGA enzyme with comparable activity in rat, dog, and mouse. Subchronic administration of MK-8719 significantly increased O-protein levels in brain tissue and reduced the formation of pathological tau species in Tg4510 mouse brain. Reduction of pathological tau accumulation in Tg4510 mice was accompanied by reductions in neurodegeneration, including reduced inflammatory marker expression, attenuation of brain weight loss, and attenuation of forebrain volume loss. In a Phase 1 study, single doses up to 1200 mg were generally well tolerated. No laboratory, ECG, or vital sign adverse experiences were observed. PET studies demonstrated that dosing of MK-8719 causes a decrease of [18F]MK-8553 binding compared to baseline, indicating target engagement of the OGA enzyme in the brain. Preclinical data demonstrate that MK-8719 significantly reduced pathological tau and neurodegeneration in Tg4510 mice. Phase 1 clinical findings support further clinical development of MK-8719 and investigation of safety and efficacy in PSP patients.

Although tau pathology, behavioral deficits, and neuronal loss are observed in patients with tauopathies, the relationship between these endpoints has not been clearly established. Here we found that rTg4510 mice, which overexpress human mutant tau in the forebrain, develop progressive age-dependent increases in locomotor activity (LMA), which correlates with neurofibrillary tangle (NFT) pathology, hyperphosphorylated tau levels, and brain atrophy. To further clarify the relationship between these endpoints, we treated the rTg4510 mice with either doxycycline to reduce mutant tau expression or an O-GlcNAcase inhibitor Thiamet G, which has been shown to ameliorate tau pathology in animal models. We found that both doxycycline and Thiamet G treatments starting at 2 months of age prevented the progression of hyperactivity, slowed brain atrophy, and reduced brain hyperphosphorylated tau. In contrast, initiating doxycycline treatment at 4 months reduced neither brain hyperphosphorylated tau nor hyperactivity, further confirming the relationship between these measures. Collectively, our results demonstrate a unique behavioral phenotype in the rTg4510 mouse model of tauopathy that strongly correlates with disease progression, and that early interventions which reduce tau pathology ameliorate the progression of the locomotor dysfunction. These findings suggest that better understanding the relationship between locomotor deficits and tau pathology in the rTg4510 model may improve our understanding of the mechanisms underlying behavioral disturbances in patients with tauopathies.

A significant hurdle in Alzheimer's disease (AD) research is the lack of translationally relevant rodent models of disease, with phenotypes similar to the pathologies found in diseased human brain. In addition, the relevance of preclinical rodent models of Alzheimer's-like pathology is poorly understood. For example, some genetically engineered mouse models (GEMMs) recapitulate either the amyloid or the tau based neuropathology observed in AD but not both features of the human disease. To better understand the relevance of preclinical models to AD, we generated genome-wide transcriptome profiles by next generation sequencing of frontal cortex from GEMMs with amyloidosis (Tg2576) or tauopathy (Tg4510) at ages spanning the onset of pathology. Significant age-dependent transcriptional changes were observed within each model. Although Tg4510 animals exhibited the greatest number and magnitude of changes, there were also many changes observed across both GEMMs. These results were compared to transcriptional modules correlating with normal aging and/or AD disease onset and progression in human brain tissue samples to determine which aspects of the human disease were present in each model. Whereas each GEMM induced gene modules associated with human aging, only Tg4510 exhibited human AD-specific signatures, suggesting that the Tg4510 mouse model may capture more aspects of biological change associated with AD than amyloidosis models. To extend these results, we generated similar transcriptional profiles from Tg4510 mice administered O-GlcNAcase (OGA) inhibitor, a treatment which reduces tau pathology and neurodegeneration in mouse. Chronic OGA inhibition reversed many transcriptional signatures specific to the progression of Tg4510 pathology and found in human AD brain. These studies highlight the utility of transcriptional profiling in determining relevance of preclinical models.

RATIONALE:The current standards of care for Alzheimer's disease, acetylcholinesterase inhibitors, have limited efficacy due to a host of mechanism-related side effects arising from indiscriminate activation of muscarinic and nicotinic receptors. The M1 muscarinic receptor is predominantly expressed in the brain in regions involved in cognition, and therefore selective activation of the M1 receptor would be expected to boost cognitive performance with reduced risk of peripheral side effects.OBJECTIVES:Here we investigated whether the selective M1 muscarinic receptor positive allosteric modulator, PQCA, improves cognitive performance and cerebral blood flow.RESULTS:PQCA attenuated a scopolamine-induced deficit in novel object recognition in rat, self-ordered spatial search in cynomolgus macaque, and the object retrieval detour task in rhesus macaque. Beneficial effects in each of these assays and species were observed at similar plasma drug concentrations. Furthermore, at similar drug concentrations that were effective in the behavioral studies, PQCA increased blood flow in the frontal cortex of mice, providing a translational biomarker that could be used to guide dose selection for clinical studies.CONCLUSIONS:These findings provide a framework for appropriately testing an M1 selective compound in patients with Alzheimer's disease.

PHARMACOLOGY Correction for ‘‘Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation,’’ by Lei Ma, Matthew Seager, Marion Wittmann, Marlene Jacobson, Denise Bickel, Maryann Burno, Keith Jones, Valerie Kuzmick Graufelds, Guangping Xu, Michelle Pearson, Alexander McCampbell, Renee Gaspar, Paul Shughrue, Andrew Danziger, Christopher Regan, Rose Flick, Danette Pascarella, Susan Garson, Scott Doran, Constantine Kreatsoulas, Lone Veng, Craig W. Lindsley, William Shipe, Scott Kuduk, Cyrille Sur, Gene Kinney, Guy R. Seabrook, and William J. Ray, which appeared in issue 37, September 15, 2009, of Proc Natl Acad Sci USA (106:15950–15955; first published August 26, 2009; 10.1073/ pnas.0900903106). The authors note that the author name Matthew Seager should have appeared as Matthew A. Seager. The online version has been corrected. The corrected author line and related footnotes appear below.

The forebrain cholinergic system promotes higher brain function in part by signaling through the M 1 muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating M 1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M 1 mAChR. BQCA reduces the concentration of ACh required to activate M 1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M 1 −/− mice demonstrates that BQCA requires M 1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M 1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M 1 , suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M 1 receptor and represents a promising therapeutic strategy for cognitive disorders.

The loss of cholinergic neurons in the basal forebrain is a hallmark of the pathophysiology of Alzheimer's disease (AD) and is believed to underlie memory loss and cognitive decline in AD patients. Based on this hypothesis, cholinesterase inhibitors have been developed and shown to be efficacious in patients. However, their efficacy is limited due to severe side effects. Therefore, novel cholinergic approaches are being broadly investigated. M1 muscarinic receptors are expressed in neurons in the cortex and hippocampus and are believed to play a central role in cognition. Therefore, we developed a selective allosteric M1 potentiator, BQCA, as a novel treatment approach for AD. Here we demonstrate that BQCA activates M1 receptors in vivo in three pharmacodynamic assays with relevance for cognition. EEG, Laser Doppler blood flow, and sleep telemetry were used to evaluate efficacy of BQCA in rodents. 1) Cognition enhancing drugs, including donepezil, have been shown to lead to an increase in theta activity in a stimulated EEG model in anesthetized rats (Kinney et al. 1998). Acute high frequency stimulation of the brainstem leads to induction of theta and gamma oscillations in the CA1 region of the hippocampus. In this model, IP dosing of 10mg/kg BQCA leads to a distinct change of EEG oscillations, mainly affecting higher EEG frequencies (20–80Hz). This effect is blocked by application of the muscarinic antagonist scopolamine (1mg/kg). 2) Non-selective muscarinic agonists have been shown to increase cerebral blood flow. We show that BQCA (10mg/kg, IV) can significantly increase cerebral blood flow measured by Laser Doppler flowmetry in anesthetized rats (20% increase) without significantly affecting mean arterial pressure. 3) Finally, we tested BQCA in a 7 day cross over sleep architecture study in awake rats. BQCA significantly affects sleep architecture at 10mg/kg IP leading to an increase in active wake and light sleep, with corresponding decreases in delta sleep and REM sleep. Taken together, these data suggest that BQCA activates M1 muscarinic receptors in the brain in a way that indicates a cognitive enhancing effect of the compound making BQCA a promising new approach for the treatment of AD.

A history of depression is a risk factor for Alzheimer's disease (AD), suggesting the possibility that antidepressants administered prophylactically might retard the disease process and preserve cognitive function. Here we report that pre-symptomatic treatment with the antidepressant paroxetine attenuates the disease process and improves cognitive performance in the 3xTgAD mouse model of AD. Five-month-old mate and female 3xTgAD and non-transgenic mice were administered either paroxetine or saline daily for 5 months. Open-field activity was tested in 7-month-old mice and performance in passive avoidance and Morris swim tasks were evaluated at 10 months. 3xTgAD mice exhibited reduced exploratory activity, increased transfer latency in the passive avoidance test and impaired performance in the Morris spatial navigation task compared to nontransgenic control mice. Paroxetine treatment ameliorated the spatial navigation deficit in 3xTgAD male and female mice, without affecting swim speed or distance traveled, suggesting a preservation of cognitive function. Levels of amyloid beta-peptide (A beta) and numbers of A beta immunoreactive neurons were significantly reduced in the hippocampus of male and female paroxetine-treated 3xTgAD mice compared to saline-treated 3xTgAD mice. Female 3xTgAD mice exhibited significantly less tau pathology in the hippocampus and amygdala compared to male 3xTgAD mice, and paroxetine lessened tau pathology in male 3xTgAD mice. The ability of a safe and effective antidepressant to suppress neuropathological changes and improve cognitive performance in a mouse model suggests that such drugs administered prophylactically might retard the development of AD in humans. (C) 2007 Elsevier Inc. All rights reserved.

Asthma is an increasingly common disorder responsible for considerable morbidity and mortality. Although obesity is a risk factor for asthma and weight loss can improve symptoms, many patients do not adhere to low calorie diets and the impact of dietary restriction on the disease process is unknown. A study was designed to determine if overweight asthma patients would adhere to an alternate day calorie restriction (ADCR) dietary regimen, and to establish the effects of the diet on their symptoms, pulmonary function and markers of oxidative stress, and inflammation. Ten subjects with BMI > 30 were maintained for 8 weeks on a dietary regimen in which they ate ad libitum every other day, while consuming less than 20% of their normal calorie intake on the intervening days. At baseline, and at designated time points during the 8-week study, asthma control, symptoms, and Quality of Life questionnaires (ACQ, ASUI, mini-AQLQ) were assessed and blood was collected for analyses of markers of general health, oxidative stress, and inflammation. Peak expiratory flow (PEF) was measured daily on awakening. Pre-and postbronchodilator spirometry was obtained at baseline and 8 weeks. Nine of the subjects adhered to the diet and lost an average of 8% of their initial weight during the study. Their asthma-related symptoms, control, and QOL improved significantly, and PEF increased significantly, within 2 weeks of diet initiation; these changes persisted for the duration of the study. Spirometery was unaffected by ADCR. Levels of serum beta-hydroxybutyrate were increased and levels of leptin were decreased on CR days, indicating a shift in energy metabolism toward utilization of fatty acids and confirming compliance with the diet. The improved clinical findings were associated with decreased levels of serum cholesterol and triglycerides, striking reductions in markers of oxidative stress (8-isoprostane, nitrotyrosine, protein carbonyls, and 4-hydroxynonenal adducts), and increased levels of the antioxidant uric acid. Indicators of inflammation, including serum tumor necrosis factor-alpha and brain-derived neurotrophic factor, were also significantly decreased by ADCR. Compliance with the ADCR diet was high, symptoms and pulmonary function improved, and oxidative stress and inflammation declined in response to the dietary intervention. These findings demonstrate rapid and sustained beneficial effects of ADCR on the underlying disease process in subjects with asthma, suggesting a novel approach for therapeutic intervention in this disorder. (c) 2006 Elsevier Inc. All rights reserved.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive decline in cognitive function associated with the neuropathological hallmarks amyloid β-peptide (Aβ) plaques and neurofibrillary tangles. Because aging is the major risk factor for AD, and dietary energy restriction can retard aging processes in the brain, we tested the hypothesis that two different energy restriction regimens, 40% calorie restriction (CR) and intermittent fasting (IF) can protect against cognitive decline in the triple-transgenic mouse model of AD (3xTgAD mice). Groups of 3xTgAD mice were maintained on an ad libitum control diet, or CR or IF diets, beginning at 3 months of age. Half of the mice in each diet group were subjected to behavioral testing (Morris swim task and open field apparatus) at 10 months of age and the other half at 17 months of age. At 10 months 3xTgAD mice on the control diet exhibited reduced exploratory activity compared to non-transgenic mice and to 3xTgAD mice on CR and IF diets. Overall, there were no major differences in performance in the water maze among genotypes or diets in 10-month-old mice. In 17-month-old 3xTgAD mice the CR and IF groups exhibited higher levels of exploratory behavior, and performed better in both the goal latency and probe trials of the swim task, compared to 3xTgAD mice on the control diet. 3xTgAD mice in the CR group showed lower levels of Aβ1–40, Aβ1–42 and phospho-tau in the hippocampus compared to the control diet group, whereas Aβ and phospho-tau levels were not decreased in 3xTgAD mice in the IF group. IF may therefore protect neurons against adverse effects of Aβ and tau pathologies on synaptic function. We conclude that CR and IF dietary regimens can ameliorate age-related deficits in cognitive function by mechanisms that may or may not be related to Aβ and tau pathologies.