Previous amyloid models have largely focused on empirically describing soluble CSF biomarkers from clinical programs or have integrated the biological steps of amyloid in theoretical models informed by literature values and/or animal data. In this analysis, the BACE inhibitor program at Merck was leveraged to fully inform an integrated mechanistic amyloid pathway model based on clinical data alone. Serial CSF biomarker data (Aβ40, Aβ42, sAPPβ, Aβ oligomers (AβO), infused 13C leucine) collected in healthy volunteers or Alzheimers (AD) patients via lumbar catherization in 3 Phase I studies for verubecestat (MK-8931) and one Phase I study each for MK-8511 and MK-8277 were integrated into a single dataset. A mechanistic model describing the Aβ pathways in brain from precursor APP pool to monomeric forms and to aggregate forms was developed, including the transit to CSF (Figure 1). BACE inhibition was added as EMAX term acting on the initial cleavage step of APP. Plaque formation was added to the model as a slow build-up process from AβO. The model provided a good simultaneous description of all study results, needing only to account for different baseline levels in the studies as well as separate IC50 parameters for each compound (4.3, 9.7, 11.9 nM for MK-8931, MK-8277, and MK-8511). This suggests that the model captures the underlying system of pathways well. No significant differences in IC50 or EMAX between AD patients and healthy subjects were found in the analysis, and the extent of the inhibition (EMAX) approached 100 % (98 %). Oligomeric Aβ equilibrated rapidly with monomers, while plaque turnover was slow.

β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) is required for the production of β‐amyloid peptides, which are implicated in the etiology of Alzheimer's disease. The safety and pharmacokinetics of the BACE1 inhibitor verubecestat have previously been studied in young adults aged 19–45 years. In this randomized, placebo‐controlled, phase I study (protocol MK‐8931‐006), we investigated the safety, tolerability, and pharmacokinetics of a single dose (100 mg) or multiple doses (30, 80, and 120 mg) once daily for 28 days of verubecestat in healthy elderly subjects. Safety end points were assessed at baseline and during the duration of the study period and indicated that verubecestat was generally well tolerated. Verubecestat pharmacokinetics were similar between healthy elderly male and female subjects and similar to those reported in healthy young males in previous studies. These data supported subsequent studies to assess the potential efficacy of verubecestat in subjects with Alzheimer's disease.

18F-MK-6240 (18F-labeled 6-(fluoro)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine) is a highly selective, subnanomolar-affinity PET tracer for imaging neurofibrillary tangles (NFTs). Plasma kinetics, brain uptake, and preliminary quantitative analysis of 18F-MK-6240 in healthy elderly (HE) subjects, subjects with clinically probable Alzheimer disease (AD), and subjects with amnestic mild cognitive impairment were characterized in a study that is, to our knowledge, the first to be performed on humans. Methods: Dynamic PET scans of up to 150 min were performed on 4 cognitively normal HE subjects, 4 AD subjects, and 2 amnestic mild cognitive impairment subjects after a bolus injection of 152–169 MBq of 18F-MK-6240 to evaluate tracer kinetics and distribution in brain. Regional SUV ratio (SUVR) and distribution volume ratio were determined using the cerebellar cortex as a reference region. Total distribution volume was assessed by compartmental modeling using radiometabolite-corrected input function in a subgroup of 6 subjects. Results: 18F-MK-6240 had rapid brain uptake with a peak SUV of 3–5, followed by a uniformly quick washout from all brain regions in HE subjects; slower clearance was observed in regions commonly associated with NFT deposition in AD subjects. In AD subjects, SUVR between 60 and 90 min after injection was high (approximately 2–4) in regions associated with NFT deposition, whereas in HE subjects, SUVR was approximately 1 across all brain regions, suggesting high tracer selectivity for binding NFTs in vivo. 18F-MK-6240 total distribution volume was approximately 2- to 3-fold higher in neocortical and medial temporal brain regions of AD subjects than in HE subjects and stabilized by 60 min in both groups. Distribution volume ratio estimated by the Logan reference tissue model or compartmental modeling correlated well (R2 > 0.9) to SUVR from 60 to 90 min for AD subjects. Conclusion: 18F-MK-6240 exhibited favorable kinetics and high binding levels to brain regions with a plausible pattern for NFT deposition in AD subjects. In comparison, negligible tracer binding was observed in HE subjects. This pilot study suggests that simplified ratio methods such as SUVR can be used to quantify NFT binding. These results support further clinical development of 18F-MK-6240 for potential application in longitudinal studies.

β‐site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of β‐amyloid (Aβ) peptides and is considered a potential treatment target for Alzheimer's disease (AD). To support Japan's participation in the global clinical development program, we characterized the safety, pharmacokinetics (PKs), and pharmacodynamics of the BACE1 inhibitor verubecestat (MK‐8931) in 24 healthy Japanese adults in a two‐part, single‐center, randomized, placebo‐controlled phase I trial (protocol MK‐8931‐007) and compared the results with historical data from non‐Japanese subjects. Both single (20, 100, and 450 mg) and multiple (80 and 150 mg once daily for 14 days) doses of verubecestat were well tolerated. Verubecestat's PK profile was similar in Japanese and non‐Japanese subjects. Verubecestat also reduced mean cerebrospinal fluid concentrations of the Aβ proteins Aβ40, Aβ42, and soluble β fragment of amyloid precursor protein; the level of reduction was comparable between Japanese and non‐Japanese subjects. These results support the continued global development of verubecestat as a potential disease‐modifying agent for Japanese and non‐Japanese subjects who are at risk for developing AD.

One of the main approaches for a disease-modifying therapy in Alzheimer's disease (AD) today, is therapeutic intervention in the amyloidogenic APP processing pathway, either via reduction in the production of Aβ monomeric precursors (secretase inhibition) or via facilitation of Aβ peptide clearance/neutralization (immunotherapy). The novel BACE inhibitor, verubecestat, was recently shown to produce a dose-dependent reduction of several APP pathway components—Aβ40, Aβ42 and sAPPβ—in healthy volunteer and AD patient CSF (Kennedy, 2016 Sci. Transl. Med. 8:363). As Aβ oligomers (AβO) embody a neurotoxic intermediary APP derivative en route to the deposition of plaques in AD brain, the present study examined whether acute inhibition of BACE would also alter AβO pool in humans. Specifically, we evaluated the pharmacodynamic (PD) response of toxic AβO, and their relationship to the response of Aβ40, in healthy volunteers after acute administration of an investigational BACE inhibitor molecule. Serial CSF samples were collected in 21 healthy males following single dose administrations of placebo, 10, 60 or 800 mg of an investigational BACE inhibitor. The PD effects in the subjects were evaluated by quantifying the levels of AβO and Aβ40 over 36 hours post-acute administration. Immunoassay-based analysis of AβO was performed as previously described (Savage, 2014 J. Neurosci. 34(8):2884–97). Robust time- and dose-dependent reductions of AβO, with similar concomitant drop of Aβ40 in the CSF of BACE inhibitor-treated subjects, were observed, reaching ∼90% relative to the baseline. These were significant for all summary measure responses tested: time-weighted averages, minimum values, concentration at three different time points, as well as time to reach minimum values. Finally, the kinetics and magnitude of response of oligomers compared with those of monomers, suggesting a great level of cooperativity between Aβ species. Together, these findings in healthy volunteers demonstrate that acute BACE inhibition reduced AβO with a similar rate and extent as monomer, suggesting potential further utility of this therapeutic approach. Future studies will examine the correlation of the magnitude and kinetics of CSF Aβ oligomers with monomeric precursors in an AD cohort after administration of secretase therapy.

β-Amyloid (Aβ) peptides are thought to be critically involved in the etiology of Alzheimer’s disease (AD). The aspartyl protease β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is required for the production of Aβ, and BACE1 inhibition is thus an attractive target for the treatment of AD. We show that verubecestat (MK-8931) is a potent, selective, structurally unique BACE1 inhibitor that reduced plasma, cerebrospinal fluid (CSF), and brain concentrations of Aβ40, Aβ42, and sAPPβ (a direct product of BACE1 enzymatic activity) after acute and chronic administration to rats and monkeys. Chronic treatment of rats and monkeys with verubecestat achieved exposures >40-fold higher than those being tested in clinical trials in AD patients yet did not elicit many of the adverse effects previously attributed to BACE inhibition, such as reduced nerve myelination, neurodegeneration, altered glucose homeostasis, or hepatotoxicity. Fur hypopigmentation was observed in rabbits and mice but not in monkeys. Single and multiple doses were generally well tolerated and produced reductions in Aβ40, Aβ42, and sAPPβ in the CSF of both healthy human subjects and AD patients. The human data were fit to an amyloid pathway model that provided insight into the Aβ pools affected by BACE1 inhibition and guided the choice of doses for subsequent clinical trials.

Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer's disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aβ levels in rats and nonhuman primates and CSF Aβ levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinane dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.

It remains an open question whether the failed efficacy trials for the gamma secretase inhibitors (semagacestat and avagacestat) and, at most, modest findings for antibodies targeting soluble Aβ (solanezumab) demonstrate that the amyloid hypothesis has been disproven. A model-based analysis was conducted to characterize the degree of modulation of Aβ tested in these trials to inform this question. A semi-mechanistic mathematical pharmacokinetic/pharmacodynamic model describing inhibition of brain Aβ production and distribution to CSF previously developed to describe CSF Aβ biomarker data from the BACE inhibitor verubecestat (MK-8931) Phase 1 program was extended to semagacestat, avagacestat, and solanezumab. Clinical data on CSF Aβ levels and drug exposure over time were digitized from the public domain. The model-estimated median steady-state % reduction (averaged over the dosing interval) in brain Aβ40 was 29.5%, 14.8%, 70.8%, and 86.8% for daily administered 50 mg avagacestat, 140 mg semagacestat, 12 mg and 40 mg verubecestat. Thus the two gamma secretase inhibitor trials likely tested a degree of Aβ reduction that was less than that associated with human genomic evidence (∼50% Aβ increase with early onset due to trisomy 21 and 40% Aβ reduction with protective BACE1 mutant). Given the genomic alterations are present at birth, it is likely that a therapeutic intervention would require a greater level of perturbation to be effective. Alterations in free and total Aβ in plasma and CSF with solanezumab were well described by a model with slowed clearance of the Aβ-mAb complex and suggested limited CNS penetration and modulation of CSF Aβ. Overall, these results indicate that the completed trials did not robustly test the amyloid hypothesis. Ongoing verubecestat trials of 12 and 40 mg are testing a substantially greater reduction in brain production of Aβ and therefore will constitute a more robust test of the amyloid hypothesis.

Dementia is often characterized as being caused by one of several major diseases, such as Alzheimer's disease (AD), cerebrovascular disease, Lewy body disease, or a frontotemporal degeneration. Failure to acknowledge that more than one entity may be present precludes attempts to understand interactive relationships. The clinicopathological studies of dementia demonstrate that multiple pathologic processes often coexist. How overlapping pathologic findings affect the diagnosis and treatment of clinical AD and other dementia phenotypes was the topic taken up by the Alzheimer's Association's Research Roundtable in October 2014. This review will cover the neuropathologic basis of dementia, provide clinical perspectives on multiple pathologies, and discuss therapeutics and biomarkers targeting overlapping pathologies and how these issues impact clinical trials.High prevalence of multiple pathologic findings among individuals with clinical diagnosis of AD suggests that new treatment strategies may be needed to effectively treat AD and other dementing illnesses.

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.

Orexin neuropeptides regulate sleep/wake through orexin receptors (OX1R, OX2R); OX2R is the predominant mediator of arousal promotion. The potential for single OX2R antagonism to effectively promote sleep has yet to be demonstrated in humans. MK-1064 is an OX2R-single antagonist. Preclinically, MK-1064 promotes sleep and increases both rapid eye movement (REM) and non-REM (NREM) sleep in rats at OX2R occupancies higher than the range observed for dual orexin receptor antagonists. Similar to dual antagonists, MK-1064 increases NREM and REM sleep in dogs without inducing cataplexy. Two Phase I studies in healthy human subjects evaluated safety, tolerability, pharmacokinetics and sleep-promoting effects of MK-1064, and demonstrated dose-dependent increases in subjective somnolence (via Karolinska Sleepiness Scale and Visual Analogue Scale measures) and sleep (via polysomnography), including increased REM and NREM sleep. Thus, selective OX2R antagonism is sufficient to promote REM and NREM sleep across species, similarly to that seen with dual orexin receptor antagonism.

The peripheral sink hypothesis contends that amyloid targeted therapies that reduce or sequester plasma Aβ without direct brain effects can still meaningfully reduce amyloid in brain by creating a sink conditions in plasma that draws Aβ from the CNS. The feasibility of this mechanism within the constraints of human physiology and well established amyloid pathway features was explored using theoretical models. An analytic solution to a two compartment (CNS and periphery) production, distribution, and elimination model was developed to approximate potential amyloid relationships. Three key constraints were applied: 1) baseline 20-fold concentration gradient (CNS-to-plasma) for Aβ; 2) baseline rapid plasma elimination rate (4 min half-life); and 3) rate of Aβ plasma-to-CNS transport cannot exceed blood flow rate to the brain. Model exploration revealed that the rate of return of Aβ from plasma-to-CNS was the key parameter constraining whether sink conditions could be created. If the 20-fold concentration gradient was derived from an equilibrium balance of plasma-to-CNS versus CNS-to-plasma transport, then it was possible to create a sink in plasma by removal of plasma Aβ that would draw out CNS Aβ. If instead the gradient derives from rate limitation of CNS-to-plasma transport (with slower reverse transport) then removal of plasma Aβ has no meaningful influence on CNS Aβ. Under conditions of perfect extraction of Aβ from plasma by brain, the maximum reduction in CNS Aβ was only 10% of that in plasma due to limitation of blood flow to the brain. For more plausible extraction ratio, therapies acting on peripheral Aβ alone have negligible effect on CNS Aβ. Additionally, rodent experiments in which CNS and peripheral Aβ were selectively modulated did not support the peripheral sink hypothesis, in agreement with the predictions of the model. These results suggest that the peripheral sink hypothesis is physiologically implausible.

INTRODUCTION:Amyloid-β (Aβ) has been investigated as a diagnostic biomarker and therapeutic drug target. Recent studies found that cerebrospinal fluid (CSF) Aβ fluctuates over time, including as a diurnal pattern, and increases in absolute concentration with serial collection. It is currently unknown what effect differences in CSF collection methodology have on Aβ variability. In this study, we sought to determine the effect of different collection methodologies on the stability of CSF Aβ concentrations over time.METHODS:Grouped analysis of CSF Aβ levels from multiple industry and academic groups collected by either lumbar puncture (n=83) or indwelling lumbar catheter (n=178). Participants were either placebo or untreated subjects from clinical drug trials or observational studies. Participants had CSF collected by lumbar puncture or lumbar catheter for quantitation of Aβ concentration by enzyme linked immunosorbent assay. Data from all sponsors was converted to percent of the mean for Aβ40 and Aβ42 for comparison. Repeated measures analysis of variance was performed to assess for factors affecting the linear rise of Aβ concentrations over time.RESULTS:Analysis of studies collecting CSF via lumbar catheter revealed tremendous inter-subject variability of Aβ40 and Aβ42 as well as an Aβ diurnal pattern in all of the sponsors' studies. In contrast, Aβ concentrations from CSF samples collected at two time points by lumbar puncture showed no significant differences. Repeated measures analysis of variance found that only time and draw frequency were significantly associated with the slope of linear rise in Aβ40 and Aβ42 concentrations during the first 6 hours of collection.CONCLUSIONS:Based on our findings, we recommend minimizing the frequency of CSF draws in studies measuring Aβ levels and keeping the frequency standardized between experimental groups. The Aβ diurnal pattern was noted in all sponsors' studies and was not an artifact of study design. Averaging Aβ concentrations at each time point is recommended to minimize the effect of individual variability. Indwelling lumbar catheters are an invaluable research tool for following changes in CSF Aβ over 24-48 hours, but factors affecting Aβ concentration such as linear rise and diurnal variation need to be accounted for in planning study designs.