Susanna Eketjäll

Discovery of AZD3839, a Potent and Selective BACE1 Inhibitor Clinical Candidate for the Treatment of Alzheimer Disease

Background: BACE1 inhibitors target the first step in Aβ formation and are tractable drugs for halting disease progression in Alzheimer disease. Results: AZD3839 is a novel BACE1 inhibitor that effectively reduces brain and CSF Aβ levels in several preclinical species. Conclusion: Based on the preclinical profile, AZD3839 was progressed into Phase I. Significance: AZD3839 may have disease-modifying potential in the treatment of Alzheimer disease. β-Site amyloid precursor protein cleaving enzyme1 (BACE1) is one of the key enzymes involved in the processing of the amyloid precursor protein (APP) and formation of amyloid β peptide (Aβ) species. Because cerebral deposition of Aβ species might be critical for the pathogenesis of Alzheimer disease, BACE1 has emerged as a key target for the treatment of this disease. Here, we report the discovery and comprehensive preclinical characterization of AZD3839, a potent and selective inhibitor of human BACE1. AZD3839 was identified using fragment-based screening and structure-based design. In a concentration-dependent manner, AZD3839 inhibited BACE1 activity in a biochemical fluorescence resonance energy transfer (FRET) assay, Aβ and sAPPβ release from modified and wild-type human SH-SY5Y cells and mouse N2A cells as well as from mouse and guinea pig primary cortical neurons. Selectivity against BACE2 and cathepsin D was 14 and >1000-fold, respectively. AZD3839 exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in mouse, guinea pig, and non-human primate. Pharmacokinetic/pharmacodynamic analyses of mouse and guinea pig data showed a good correlation between the potency of AZD3839 in primary cortical neurons and in vivo brain effects. These results suggest that AZD3839 effectively reduces the levels of Aβ in brain, CSF, and plasma in several preclinical species. It might, therefore, have disease-modifying potential in the treatment of Alzheimer disease and related dementias. Based on the overall pharmacological profile and its drug like properties, AZD3839 has been progressed into Phase 1 clinical trials in man.

Design and synthesis of β-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors with in vivo brain reduction of β-amyloid peptides.

The evaluation of a series of aminoisoindoles as β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimers disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aβ40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 μM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of β-amyloid peptides in mouse brain following oral dosing.

New Aminoimidazoles as β-Secretase (BACE-1) Inhibitors Showing Amyloid-β (Aβ) Lowering in Brain

Amino-2H-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimers disease. Synthetic methods, crystal structures, and structure-activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (S)-1m was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (S)-1m, a concentration and time dependent decrease in Aβ40 and Aβ42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aβ was 40-50%, 1.5 h after oral dosing (100 μmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo.

AZ-4217: a high potency BACE inhibitor displaying acute central efficacy in different in vivo models and reduced amyloid deposition in Tg2576 mice.

Aβ, the product of APP (amyloid precursor protein), has been implicated in the pathophysiology of Alzheimers disease (AD). β-Site APP cleaving enzyme1 (BACE1) is the enzyme initiating the processing of the APP to Aβ peptides. Small molecule BACE1 inhibitors are expected to decrease Aβ-peptide generation and thereby reduce amyloid plaque formation in the brain, a neuropathological hallmark of AD. BACE1 inhibition thus addresses a key mechanism in AD and its potential as a therapeutic target is currently being addressed in clinical studies. Here, we report the discovery and the pharmacokinetic and pharmacodynamic properties of BACE1 inhibitor AZ-4217, a high potency compound (IC50 160 pm in human SH-SY5Y cells) with an excellent in vivo efficacy. Central efficacy of BACE1 inhibition was observed after a single dose in C57BL/6 mice, guinea pigs, and in an APP transgenic mouse model of cerebral amyloidosis (Tg2576). Furthermore, we demonstrate that in a 1 month treatment paradigm BACE1 inhibition of Aβ production does lower amyloid deposition in 12-month-old Tg2576 mice. These results strongly support BACE1 inhibition as concretely impacting amyloid deposition and therefore potentially an important approach for therapeutic intervention in AD.

AZD3293: A Novel, Orally Active BACE1 Inhibitor with High Potency and Permeability and Markedly Slow Off-Rate Kinetics

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 amyloid-β peptide (Aβ) concentrations, plays a key role in the pathogenesis of Alzheimer’s disease (AD). Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid-β protein precursor (AβPP) to Aβ peptides, with the soluble N terminal fragment of AβPP (sAβPPβ) as a direct product, and BACE1 inhibition is an attractive target for therapeutic intervention to reduce the production of Aβ. Here, we report the in vitro and in vivo pharmacological profile of AZD3293, a potent, highly permeable, orally active, blood-brain barrier (BBB) penetrating, BACE1 inhibitor with unique slow off-rate kinetics. The in vitro potency of AZD3293 was demonstrated in several cellular models, including primary cortical neurons. In vivo in mice, guinea pigs, and dogs, AZD3293 displayed significant dose- and time-dependent reductions in plasma, cerebrospinal fluid, and brain concentrations of Aβ40, Aβ42, and sAβPPβ. The in vitro potency of AZD3293 in mouse and guinea pig primary cortical neuronal cells was correlated to the in vivo potency expressed as free AZD3293 concentrations in mouse and guinea pig brains. In mice and dogs, the slow off-rate from BACE1 may have translated into a prolongation of the observed effect beyond the turnover rate of Aβ. The preclinical data strongly support the clinical development of AZD3293, and patients with AD are currently being recruited into a combined Phase 2/3 study to test the disease-modifying properties of AZD3293.

Revisiting the peripheral sink hypothesis: inhibiting BACE1 activity in the periphery does not alter β‐amyloid levels in the CNS

Aggregation of amyloid beta (Aβ) peptides and the subsequent neural plaque formation is a central aspect of Alzheimers disease. Various strategies to reduce Aβ load in the brain are therefore intensely pursued. It has been hypothesized that reducing Aβ peptides in the periphery, that is in organs outside the brain, would be a way to diminish Aβ levels and plaque load in the brain. In this report, we put this peripheral sink hypothesis to test by investigating how selective inhibition of Aβ production in the periphery using a β‐secretase (BACE)1 inhibitor or reduced BACE1 gene dosage affects Aβ load in the brain. Selective inhibition of peripheral BACE1 activity in wild‐type mice or mice over‐expressing amyloid precursor protein (APPswe transgenic mice; Tg2576) reduced Aβ levels in the periphery but not in the brain, not even after chronic treatment over several months. In contrast, a BACE1 inhibitor with improved brain disposition reduced Aβ levels in both brain and periphery already after acute dosing. Mice heterozygous for BACE1, displayed a 62% reduction in plasma Aβ40, whereas brain Aβ40 was only lowered by 11%. These data suggest that reduction of Aβ in the periphery is not sufficient to reduce brain Aβ levels and that BACE1 is not the rate‐limiting enzyme for Aβ processing in the brain. This provides evidence against the peripheral sink hypothesis and suggests that a decrease in Aβ via BACE1 inhibition would need to be carried out in the brain.

TREM2 shedding by cleavage at the H157‐S158 bond is accelerated for the Alzheimer's disease‐associated H157Y variant

We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2‐expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N‐terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157‐S158 peptide bond for both wild‐type and H157Y human TREM2 and for the wild‐type murine orthologue. Crucially, we also show that the Alzheimers disease‐associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat‐ and ADAM10‐siRNA‐independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimers and other neurological diseases.

BACE1 Inhibitor Lanabecestat (AZD3293) in a Phase 1 Study of Healthy Japanese Subjects: Pharmacokinetics and Effects on Plasma and Cerebrospinal Fluid Aβ Peptides

Lanabecestat (AZD3293; LY3314814) is an orally active potent inhibitor of human β‐secretase 1 in clinical development for the treatment of Alzheimer disease. In this first Japanese clinical study for an Alzheimer disease intervention to include cerebrospinal fluid (CSF) sampling in Japanese elderly healthy subjects, we report the pharmacokinetics and effects on plasma and CSF amyloid‐β (Aβ) peptides of lanabecestat in a phase 1 study involving 40 healthy Japanese subjects (NCT02005211). No safety and tolerability concerns were identified in healthy Japanese subjects exposed to lanabecestat up to the highest doses given, which is consistent with observations in a US phase 1 study of lanabecestat. Exposure to lanabecestat was similar for young and elderly subjects and increased in a dose‐dependent manner. For elderly subjects, plasma lanabecestat half‐life after multiple dosing was 12 to 17 hours (on days 10 and 14). Robust plasma and CSF Aβ peptide reductions were also seen at all doses, with CSF Aβ42 concentrations reduced by 63% and 79% in the 15‐ and 50‐mg lanabecestat groups, respectively. CSF soluble amyloid‐β precursor protein β also decreased following lanabecestat treatment. Suppression of CSF Aβ peptides was similar in elderly healthy Japanese subjects and US patients with mild to moderate Alzheimer disease. Lanabecestat is a promising potentially disease‐modifying treatment in phase 3 development for patients with early Alzheimer disease.

Modeling of age-dependent amyloid accumulation and γ-secretase inhibition of soluble and insoluble Aβ in a transgenic mouse model of amyloid deposition

According to the “amyloid hypothesis,” accumulation of amyloid beta (Aβ) peptides in the brain is linked to the development of Alzheimers disease. The aims of this investigation were to develop a model for the age‐dependent amyloid accumulation and to quantify the age‐ and treatment‐duration‐dependent efficacy of the γ‐secretase inhibitor MRK‐560 in the Tg2576 transgenic mouse model of amyloid deposition. Soluble and insoluble Aβ40 and Aβ42 brain concentrations were compiled from multiple naïve, vehicle, and MRK‐560‐treated animals. The age of Tg2576 mice in the studies ranged between 3.5 and 26 months. Single doses of MRK‐560 inhibited soluble Aβ40 levels in animals up to 9 months old. In contrast, MRK‐560 did not cause significant acute effects on soluble Aβ40 levels in animals older than 13 months. Absolute levels of Aβ variants increased exponentially over age and reached a plateau at ~20 months. In the final model, it was assumed that MRK‐560 inhibited the Aβ production rate with an Aβ level‐dependent IC50.The age‐dependent increase in Aβ levels was best described by a logistic model that stimulated the production rate of soluble Aβ. The increase in insoluble Aβ was defined as a function of soluble Aβ by using a scaling factor and a different turnover rate. The turnover half‐life for insoluble Aβ was estimated at 30 days, explaining that at least a 4‐week treatment in young animals was required to demonstrate a reduction in insoluble Aβ. Taken together, the derived knowledge could be exploited for an improved design of new experiments in Tg2576 mice.

AZD3293, a potent and selective orally active, brain-permeable BACE1 inhibitor

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.