Theresa A. Day

The Potent BACE1 Inhibitor LY2886721 Elicits Robust Central Aβ Pharmacodynamic Responses in Mice, Dogs, and Humans

BACE1 is a key protease controlling the formation of amyloid β, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimers disease (AD). Therefore, the development of potent and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academia and industry. Herein, we report the nonclinical and early clinical development of LY2886721, a BACE1 active site inhibitor that reached phase 2 clinical trials in AD. LY2886721 has high selectivity against key off-target proteases, which efficiently translates in vitro activity into robust in vivo amyloid β lowering in nonclinical animal models. Similar potent and persistent amyloid β lowering was observed in plasma and lumbar CSF when single and multiple doses of LY2886721 were administered to healthy human subjects. Collectively, these data add support for BACE1 inhibition as an effective means of amyloid lowering and as an attractive target for potential disease modification therapy in AD.

Up-regulation of astrocyte metabotropic glutamate receptor 5 by amyloid-β peptide

The effects of amyloid-beta peptide (Aβ) on astrocyte responses to activation of mGlu5 receptors have been investigated using calcium imaging. Pre-incubation with Aβ1-40 peptide for up to 72 h produced a time- and concentration-dependent 2-4 fold enhancement in the magnitude of the intracellular calcium mobilization response to the group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG). In contrast, pre-treatment with Aβ1-40 did not alter the calcium responses induced by other G protein coupled- or ion channel-receptors. Aβ 1-40-enhanced DHPG responses were blocked by the mGlu5 antagonist MPEP but not by inhibitors of voltage dependent calcium channels or by the AMPA/KA receptor antagonist CNQX. Up-regulation of mGlu5 coupled responses was associated with significant increases in astrocyte mGlu5 receptor-mRNA and-protein expression after preincubation with Aβ . The changes observed in vitro were consistent with results obtained from human Alzheimers disease (AD) patients.Immunostaining for mGlu5 receptors was increased on astrocytes which were colocalized with Aβ plaques in hippocampal tissue from AD patients compared to age-matched controls. These results suggest that modulation of mGlu5 receptors in astrocytes could be an important mechanism in determining the progression of pathology in AD.

Development of Homogeneous 384-Well High-Throughput Screening Assays for Aβ1-40 and Aβ1-42 Using AlphaScreen™ Technology

Amyloid beta (Aβ) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimers disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimers disease research. The current most established assay for analysis of Aβ peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Aβ1-42 and Aβ1-40 using AlphaScreen™ technology. The assays are capable of detecting Aβ peptides at concentrations <2 pg/mL and, using a final assay volume of 20 µL, routinely generate Z′ values >0.85. The AlphaScreen™ format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen™ technology permits robust, efficient, and cost-effective quantitation of Aβ peptides. (Journal of Biomolecular Screening 2008:101-111)

MEASUREMENT OF ENDOGENOUS MOUSE TAU IN CEREBROSPINAL FLUID FROM AGED PDAPP MICE FOLLOWING TREATMENT WITH AB-LOWERING COMPOUNDS

Background:Dementia is usually attributable to one or a combination of neuropathologic abnormalities, including Alzheimer amyloid plaques and neurofibrillary tangles. Most demented individuals have at least two types of dementia-related abnormality. Older decedents without abnormalities may be viewed as showing resistance to the relevant pathogenic processes. Others with brain or cognitive reserves may maintain normal cognitive performance even when 1 or 2 types of histopathologic abnormality have developed. We hypothesize essential resilience as a third defense against established neuropathology. Methods: Analyses involved 572 autopsied HAAS participants (Japanese-American men) dying at age 80 or older whose baseline Cognitive Assessment and Screening Instrument (CASI) score had been normal (>1⁄474). End-of-life severe impairment was based on a final CASI score <60. Four overlapping reserve elements were each dichotomized as 0 or 1: (i) negligible brain atrophy, (ii) higher education, (iii) higher occupational complexity, (iv) higher adult life cognitive test scores. A neuropathologic burden index (Neurology, March 2016) was the sum of four histopathologic abnormalities, each scored 0.4 (moderate) or 1.0 (severe) for Alzheimer lesions, microinfarcts, Lewy bodies, and hippocampal sclerosis. Results:Among 159 participants with a neuropathologic burden index of zero, 145 (91%) maintained normal cognition. Among 387 with burden indices 0.4 1.8 (most with two moderate or severe lesion types), the proportions developing severe impairment fell from 49%, to 35%, to 29%, to 25%, to 13% respectively in decedents with zero, 1, 2, 3, or all 4 reserve elements. Of 26 decedents with neuropathologic burden indices >1⁄42, 22 (85%) developed severe cognitive impairment. We continue to search for factors determining essential resilience. Conclusions: In this autopsied cohort, brain and/or cognitive reserves provided a powerful attenuation of cognitive impairment attributable to a panel of neuropathological abnormalities. A hypothesized additional contribution by essential resilience remains to be demonstrated.

OPTIMIZATION OF THE PHARMACOKINETIC (PK) AND PHARMACODYNAMIC (PD) PROPERTIES OF AN ANTI-ABETA ANTIBODY M266 FAB FRAGMENT VARIANT BY SITE-SPECIFIC PEGYLATION

neuronal culture assay with an EC50of approximately 250 pM. Following acute oral treatment with 0, 0.3, 1.0, or 3.0 mg/kg LY3202626, dose-dependent reductions in Ab, sAPPb, and C99 were observed in cortex and hippocampus of PDAPPmice. In beagle dog, acute oral dosing of 1.5 mg/kg LY3202626 resulted in lowering of plasma and CSF Ab 1-x by approximately 80% at the nadir; the CSF Ab 1-x was still reduced by approximately 75% at 24 hours post-dosing. Exposure of LY3202626 in plasma and CSF correlated significantly with pharmacodynamic effects upon Ab in both PDAPP mice and beagle dogs. Conclusions:LY3202626 is a potent and selective inhibitor of the BACE1 and BACE2 enzymes. The robust in vivo effects of LY3202626 are consistent with its in vitro potency and exposure in target compartments. These data support further clinical development of LY3202626 for the treatment of AD.

Standardization of beta-amyloid reference peptides

Samantha Budd Haeberlein, Gvido Cebers, Kina H€oglund, Hugh Salter, Susanna Eketj€all, Anna Bogstedt, Tina Olsson, Robert Alexander, Michael Poole, AstraZeneca R&D, Cambridge, Massachusetts, United States; Translational Science Centre, Solna, Sweden. Contact e-mail: samantha.budd@azneuro.com Background: A growing body of pathological, biomarker, genetic and mechanistic data suggests that amyloid accumulation as a result of changes in production, processing and/or clearance of brain Ab levels plays a key role in the pathogenesis of Alzheimer’s disease (AD). G enetic mutations in APP have been linked causally to earlyonset AD, and two mutations in APP (K670N/M671L the Swedish mutation, and the A673T variant) have been associated with changes in Beta-site amyloid precursor protein cleaving enzyme1 (BACE1) activity and confer early onset AD and reduced risk for AD respectively. BACE1 is the first step in the processing of APP to Ab peptides, and its inhibition is an attractive target for therapeutic intervention to stop the production of A b.Methods:We report here the pharmacological profile of a potent and selective, orally active, brain permeable BACE1 inhibitor AZD3293. Results: The potency of AZD3293 in cellular models on secretion of Ab40 has been studied in SHSY5Y/APP cells (human neuronal cells over expressing human APPwt), N2A cells (mouse neuronal cells), primary mouse neurons and primary guinea pig neurons, using ELISA technology. Mice treated with AZD3293 as a single administration, or repeated administrations twice daily during 7 days, demonstrated a statistically significant doseand time-dependent reduction of the levels of Ab40, Ab42 and sAPPb in plasma and brain. Guinea pigs treatedwithAZD3293 as a single administration demonstrated a statistically significant doseand time-dependent reduction of the levels of Ab40, Ab42 and sAPPb in plasma, CSF and brain. In vitro potency inmouse and guinea pig primary cortical neuronal cells was strongly correlated to potency in mouse mouse and guinea pig in vivo potency.Conclusions: In conclusion, AZD3293 is a potent and selective, orally active, brain permeable BACE1 inhibitor with a promising preclinical profile for treatment of AD.

Characterization of GSAP's role in modulating APP processing

Background: Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder affecting nearly 25 million patients. It is characterized by progressive cognitive decline and eventually a debilitating dementia. Currently available pharmacologic interventions only provide symptomatic relief without halting the progression of the disease. Thus, there is an enormous medical need for novel disease-modifying therapies that target the underlying neuropathological mechanisms involved in the development of AD. Strong genetic, physiological and biochemical evidence suggests that b -amyloid (Ab) plays a key role in AD. Preventing Ab aggregation is therapeutically attractive because this process is believed to be the main pathological event, and does not interfere with the physiological role of the amyloid precursor protein (APP). Methods:We have employed a set of rationally designed non-dye compounds. The aim was to inhibit Ab1-42 oligomerization and to disaggregate pre-formed Ab1-42 oligomers. Herein, we present in vitro data of our compounds obtained by thioflavin-T (ThT) assay and other biochemical assays. In addition, we present preclinical data of our compounds (metabolic stability, P-glycoprotein mediated drug efflux, brain penetration, etc.). Selected compounds were then administered to female hAPPL mice by gavage for the duration of 4 weeks. Results: The ThT-assay, used as first screening tool, was complemented by other biochemical assays and allowed the identification of compounds with suitable inhibition of Ab1-42 aggregation and toxicity Preclinical data allowed the selection of 6 compounds with suitable brain penetration for testing in female hAPPL mice. Although all 6 compounds displayed good inhibition of Ab1-42 aggregation and suitable brain penetration properties, not all of them improved spatial cognition of female hAPPL mice in the water-maze task. In addition, some compounds displayed a reduction of soluble and insoluble Ab in brain extracts. Conclusions: We have discovered a set of small molecules which prevented/reversed the pathological toxic effect of Ab and improved memory deficits of female hAPPL mice. Thus, these compounds could be promising candidates for the treatment of AD.

P1-007: Alterations in metabotropic and ionotropic glutamate receptor expression in the APP V717F transgenic mouse model of Alzheimer’s disease

accompanied with memory impairment followed by global cognitive deficits. Accumulation of beta-amyloid peptide 42 (A 42) in the brain is widely believed to be central etiology of AD. However, the precise molecular mechanisms underlying neuronal injury and death caused by A 42 remain to be elucidated. In vitro, A 42 can form a wide array of structures, including multiple monomer conformers, various size of oligomers, A derived diffusible ligands (ADDLs), protofibrils, fibrils and spheroids. Recent research efforts have been focused on understating the relationship between these different misfolding structures and neurotoxicity and the relevance to AD pathogenesis. In previous work, we have reported that the transgenic flies expressing A 42 fused to secretion signal in neurons showed progressive amyloid deposits formation, learning defects and neurodegeneration. Our recent study using immuno-electron microscopy further confirmed that most of A 42 accumulation occurred intraneuronally. These results indicate our A 42 fly is a useful model system to study intraneuronal toxicity of A 42 in vivo.