Alzheimer's disease (AD) remains a critical unmet medical need that Eli Lilly and Company has been working to address for many years. This manuscript details the evolution of Lilly's BACE inhibitor program, highlighting the iterative synthetic strategies that enabled the advancement of multiple clinical development candidates. The work underscores how strategic retrosynthetic analysis and process optimization facilitated the translation of intricate medicinal chemistry targets into robust manufacturing processes, ultimately supporting clinical development efforts in AD despite the inherent challenges and program terminations encountered along the way.

Importance:Inhibiting O-linked N-acetylglucosaminidase (OGA) is hypothesized to slow accumulation of aggregated, hyperphosphorylated tau and neurofibrillary tangle formation. Objective:To evaluate the efficacy and safety of the potent oral OGA inhibitor ceperognastat in early symptomatic Alzheimer disease (AD). Design, Setting, and Participants:This double-blind, randomized, placebo-controlled phase 2 study was conducted at 72 sites in 5 countries from September 2021 to August 2024, with a posttreatment observational extension period through May 2025. The primary outcome population comprised participants with early symptomatic AD and biomarker evidence of tau pathology, including low to medium baseline tau levels (excluding individuals with high levels), as measured by positron emission tomography (PET) with flortaucipir F18. Intervention:Participants were randomized 1:1:1 to receive once-daily, oral ceperognastat at doses of 0.75 mg (n = 110) or 3 mg (n = 108) or placebo (n = 108). Main Outcomes and Measures:The primary outcome was change in Integrated AD Rating Scale (iADRS) score. The success criterion was a 60% or greater probability of achieving greater than or equal to 25% slower progression vs placebo using a bayesian probabilistic disease progression model. The 6 secondary outcomes were Alzheimer's Disease Assessment Scale-Cognitive Subscale, 13-item version; Alzheimer's Disease Cooperative Study-Activities of Daily Living scale; Clinical Dementia Rating-Sum of Boxes; Mini-Mental State Examination; tau PET; and volumetric magnetic resonance imaging. Results:Of the 327 randomized participants (mean age, 73.4 years; 201 [61.5%] females), 259 (87 receiving ceperognastat 0.75 mg, 86 receiving ceperognastat 3 mg, and 86 receiving placebo) with low to medium baseline tau levels were included in the primary outcome population (mean age, 74.3 years; 161 [62.2%] females). In this population, no clinically meaningful benefit vs placebo was observed for the primary end point (iADRS score) at 100 weeks: posterior mean change from baseline was -8.39 with ceperognastat 0.75 mg, -13.27 with ceperognastat 3 mg, and -10.07 with placebo. The disease progression ratio relative to placebo was 0.84 (95% credible interval [CrI], 0.66-1.04) for the ceperognastat 0.75 mg group and 1.32 (CrI, 1.10-1.58) for the ceperognastat 3 mg group, corresponding to 16% less and 32% greater progression, respectively. No benefits were demonstrated for secondary clinical end points. From baseline to 76 weeks, least-squares mean change in standardized uptake value ratio on PET showed a statistically significantly smaller increase (P = .04) vs placebo only for the ceperognastat 3 mg group in the lateral temporal lobe. Volumetric magnetic resonance imaging showed less whole brain volume loss in participants treated with ceperognastat vs placebo (43.2% [ceperognastat 0.75 mg] and 49.5% [ceperognastat 3 mg] less vs placebo; both P < .001). The ceperognastat 3 mg group had more serious (ceperognastat 0.75 mg: n = 13 [12.0%]; ceperognastat 3 mg: n = 29 [26.4%]; placebo: n = 17 [15.7%]) and severe (ceperognastat 0.75 mg: n = 7 [6.5%]; ceperognastat 3 mg: n = 15 [13.6%]; placebo: n = 7 [6.5%]) treatment-emergent adverse events. Conclusions and Relevance:Ceperognastat did not slow disease progression of early symptomatic AD. Trial Registration:ClinicalTrials.gov Identifier: NCT05063539.

INTRODUCTION:The aggregation and spread of hyperphosphorylated, pathological tau in the human brain is hypothesized to play a key role in Alzheimer's disease (AD) as well as other neurogenerative tauopathies. O-GlcNAcylation, an important post-translational modification of tau and many other proteins, is significantly decreased in brain tissue of AD patients relative to healthy controls. Increased tau O-GlcNAcylation has been shown to reduce tau pathology in mouse in vivo tauopathy models. O-GlcNAcase (OGA) catalyzes the removal of O-GlcNAc from tau thereby driving interest in OGA inhibition as a potential therapeutic approach to reduce tau pathology and slow the progression of AD. METHODS:A multidisciplinary approach was used to identify ceperognastat (LY3372689) as a potent OGA inhibitor, including an extensive discovery effort with synthetic chemistry, structure-based drug design, and in vivo OGA enzyme occupancy studies. Preclinical studies assessed the target engagement, inhibition of OGA enzyme activity, OGA enzyme occupancy, and changes in tau O-GlcNAc. Four clinical Phase 1 studies of ceperognastat in healthy participants were performed to assess clinical safety and tolerability, pharmacokinetics (PK), and enzyme occupancy. RESULTS:Ceperognastat is a potent, central nervous system (CNS)-penetrant, low-dose inhibitor of OGA, which can achieve > 95% OGA enzyme occupancy in animal and human brain. Overall, ceperognastat had an acceptable safety profile in Phase 1 clinical studies with no serious adverse events reported following single and multiple dosing. The PK, enzyme occupancy, and safety profile supported Phase 2 development of ceperognastat. DISCUSSION:Ceperognastat is an orally available, highly potent, CNS-penetrant OGA inhibitor that achieved high (> 80%) OGA enzyme occupancy and increased brain O-GlcNAc-tau preclinically. Ceperognastat demonstrated > 95% OGA enzyme occupancy in Phase 1 trials. These occupancy data informed the dose selection for the Phase 2 clinical program. Highlights:Ceperognastat is a highly potent, CNS-penetrant OGA inhibitor.Ceperognastat is both orally available and CNS-penetrant even when given at low doses.Ceperognastat can achieve > 95% OGA enzyme occupancy in the animal and human brain.Ceperognastat had an acceptable safety profile in Phase 1 clinical studies.

Inhibition of O-linked N-acetyl glucosaminidase (OGA) is hypothesized to impede the formation of hyperphosphorylated, insoluble tau aggregates and neurofibrillary tangles. LY3372689 is an orally available, potent inhibitor of OGA being tested in the ongoing PROSPECT-ALZ study. This is the first study assessing the safety and efficacy of an OGA inhibitor in individuals with early symptomatic Alzheimer’s Disease (AD). To improve efficiencies in identifying individuals with desired levels of tau pathology, PROSPECT-ALZ implemented a gated screening approach using plasma P-tau217 as an enrichment step prior to tau PET. We present the study design, findings from the screening process, and baseline characteristics of the PROSPECT-ALZ study cohort. With 68 sites across Australia, Canada, Japan, Poland, and the US, PROSPECT-ALZ is an ongoing multicenter, double-blind, placebo-controlled Phase 2 study with 1:1:1 randomization of daily LY3372689 low dose, LY3372689 high dose, or placebo. The primary objective is to evaluate the effect of LY3372689 on AD clinical progression, as assessed by the change from baseline to endpoint (76-124 weeks with common close design) on the integrated AD Ratings Scale (iADRS). Pre-screening procedures include the Mini-Mental State Examination (MMSE, scores of 22-30) and a plasma P-tau217 test to determine eligibility for further screening. Subsequent key inclusion requirements to meet were age 60-85 years, a CDR global score of 0.5 or 1 with a memory box score of ≥ 0.5, and a PET (18F-flortaucipir) scan demonstrating an intermediate or high level of aggregated brain tau by combined visual and quantitative assessment. After consent, 2180 participants entered screening. The top reason for screen failure was a P-tau217 result below the eligibility threshold. Performance of the P-tau217 test for pre-screening patient selection as well as characteristics and demographics of the screening and randomized cohorts will be presented. As blood-based biomarkers become more accessible, their use in selecting participants for clinical research is desirable. The PROSPECT-ALZ study successfully implemented plasma P-tau217 in sequence with tau PET to improve efficiency in identifying trial participants. This study of LY3372689, the first orally administered OGA inhibitor to be investigated in AD, has topline trial results anticipated in 2024.

Background: The development of beta-site amyloid-beta precursor protein cleaving enzyme (BACE) 1 inhibitors for the treatment of Alzheimer’s disease requires optimization of inhibitor potency, selectivity, and brain penetration. Moreover, there is a need for low-dose compounds since liver toxicity was found with some BACE inhibitors. Objective: To determine whether the high in vitro potency and robust pharmacodynamic effect of the BACE inhibitor LY3202626 observed in nonclinical species translated to humans. Methods: The effect of LY3202626 versus vehicle on amyloid-β (Aβ) levels was evaluated in a series of in vitro assays, as well as in in vivo and multi-part clinical pharmacology studies. Aβ levels were measured using analytical biochemistry assays in brain, plasma, and cerebrospinal fluid (CSF) of mice, dogs and humans. Nonclinical data were analyzed using an ANOVA followed by Tukey’s post hoc test and clinical data used summary statistics. Results: LY3202626 exhibited significant human BACE1 inhibition, with an IC50 of 0.615±0.101 nM in a fluorescence resonance energy transfer assay and an EC50 of 0.275±0.176 nM for lowering Aβ1–40 and 0.228±0.244 nM for Aβ1–42 in PDAPP neuronal cultures. In dogs, CSF Aβ1hboxx concentrations were significantly reduced by ∼80% at 9 hours following a 1.5 mg/kg dose. In humans, CSF Aβ1–42 was reduced by 73.1±7.96 % following administration of 6 mg QD. LY3202626 was found to freely cross the blood-brain barrier in dogs and humans. Conclusion: LY3202626 is a potent BACE1 inhibitor with high blood-brain barrier permeability. The favorable safety and pharmacokinetic/pharmacodynamic profile of LY3202626 supports further clinical development.

The beta-site APP cleaving enzyme 1, known as BACE1, has been a widely pursued Alzheimer's disease drug target owing to its critical role in the production of amyloid-beta. We have previously reported the clinical development of LY2811376 and LY2886721. LY2811376 advanced to Phase I before development was terminated due to nonclinical retinal toxicity. LY2886721 advanced to Phase II, but development was halted due to abnormally elevated liver enzymes. Herein, we report the discovery and clinical development of LY3202626, a highly potent, CNS-penetrant, and low-dose BACE inhibitor, which successfully addressed these key development challenges.

LY3372689, an OGA enzyme inhibitor, is being developed as a potential treatment for tauopathies, including Alzheimer’s disease. OGA inhibition is proposed to delay the progression of tau-related diseases by slowing the accumulation of hyper-phosphorylated, insoluble tau filaments. We report the safety and pharmacokinetics (PK) of LY3372689 after single and multiple oral doses in healthy volunteers (HV). The single ascending dose (SAD) and multiple ascending dose (MAD) studies were single center, subject- and investigator-blind, placebo-controlled and randomized. In the SAD study [NCT03819270], 6 LY3372689 dose levels up to 16 mg and placebo were evaluated. In the MAD study [NCT04106206], LY3372689 (1, 3 and 7 mg) or placebo was given once daily (QD) for 14 days. Safety was assessed by adverse events (AE), safety laboratories, electrocardiograms, vital signs, physical exams, and neurological exams. Plasma pharmacokinetics (PK) was assessed in the SAD and MAD studies. In the SAD, 23 HV (15 males, 8 females; 22 – 63 years) participated, of which 18 HV completed. In the MAD, 40 HV (5 males, 35 females; 29 – 65 years) participated in the study, of which 39 HV completed. LY3372689 was generally well tolerated up to the highest dose in each study, and no serious AE were reported. In the SAD, 40 treatment-emergent AEs (TEAEs) were reported, which were mostly mild in severity. The most common TEAEs were headache, nausea, pain in extremity, pain of skin, vessel puncture site pain, and limb discomfort. In the MAD, 42 TEAEs were reported, all of which were mild in severity. The most reported TEAE was headache. In both studies, there were no clinically significant changes in safety laboratories, including markers of inflammation, muscle injury, hormones and hepatoxicity. Following QD dosing of LY3372689, the t max and t 1/2 was about 1 hour and 6 hours, respectively, and LY3372689 exposure accumulation was minimal. Renal clearance was not a major contributor of LY3372689 elimination. LY3372689 demonstrated an acceptable safety and PK profile following single and multiple doses of LY3372689 in HV. These results support investigation of LY3372689 in efficacy trials for tauopathies and help support dose selection for those trials.

LY3372689, an O-GlcNAcase (OGA) enzyme inhibitor, is being developed as a potential treatment of tauopathies, including Alzheimer’s disease. OGA inhibition is proposed to delay the progression of tau-related diseases by slowing the accumulation of hyper-phosphorylated, insoluble tau filaments. We report on single dose (SD) and multiple dose (MD) clinical studies testing the effect of LY3372689 on brain OGA enzyme occupancy (EO). A positron emission tomography (PET) radioligand, 18 F-LY3316612, was used to assess brain OGA EO in healthy volunteers (HV). In the SD study [NCT03944031], 0.25, 1 and 5 mg LY3372689 were evaluated across 4 cohorts (N = 4 HV per cohort). Each HV had PET scans at baseline and two post-dose intervals. Post-dose scans occurred at 2 and 24 hours at 0.25, 1, and 5 mg, with additional scans for 1 mg at 30 and 54 hours. The MD study [NCT04392271] consisted of 1 cohort (N = 4 HV) given 1 mg LY3372689 once daily for 14 days. Each HV had a baseline PET scan, and PET scans at 24 hours post-dose after the 1 st and 14 th administration of LY3372689. Plasma pharmacokinetics was assessed in these studies. In the SD study, the mean brain OGA EO at 5 mg was 98% at 2 hours and 93% at 24 hours. At 1 mg, the mean EO was 97% at 2 hours, 81% at 24 hours, 68% at 30 hours, and 30% at 54 hours. The EO at 0.25 mg was lower at 2 hours (26%) compared to 24 hours (46%). The E max and EC 50 values were estimated to be 97% and 0.1 ng/mL, respectively. In the MD study, the OGA EO at 24 hours after the 1 st and 14 th administration of 1 mg LY3372689 was 84%. PET studies in healthy volunteers demonstrated that LY3372689 can achieve high brain target occupancy of the OGA enzyme. Brain OGA EO was maintained after multiple dosing, supporting the durability of target engagement for longer clinical trials. The human PET data will be used to support LY3372869 dose selection for efficacy trials in tauopathies.

Herein we report a summary of the synthetic development of LY3202626 from the initial discovery route to a final route that was scaled to make 150 kg. Key developments include the use of a [3 + 2] cyclization to set the cis ring junction of the formed isoxazoline, a one-pot thiazine formation, and three different ways to install the aniline: (1) Cu-catalyzed azide coupling and reduction, (2) nitration and reduction, and (3) Buchwald coupling with acetamide.

LY3372689, an O‐GlcNAcase (OGA) enzyme inhibitor, is being developed as a potential treatment of tauopathies, including Alzheimer’s disease. OGA inhibition is proposed to delay the progression of tau‐related diseases by slowing the accumulation of hyper‐phosphorylated, insoluble tau filaments. Herein, we report on nonclinical and clinical studies that assessed the effect of LY3372689 on brain OGA enzyme occupancy (EO).

AbstractBackgroundLY3372689, an O‐GlcNAcase (OGA) enzyme inhibitor, is being developed as a potential treatment for tauopathies, including Alzheimer’s disease. OGA inhibition is proposed to delay the progression of tau‐related diseases by slowing the accumulation of hyper‐phosphorylated, insoluble tau filaments. Herein we report the first clinical study that assessed safety and pharmacokinetics (PK) of LY3372689 after single oral doses in healthy volunteers (HV).MethodThe phase 1 clinical trial (NCT03819270) was a single ascending dose, randomized, crossover, placebo controlled, investigator‐ and subject‐blind study. Each HV received up to 3 escalating single doses of LY3372689 or placebo over 3 study periods with an approximately 7‐day follow‐up period after each dose, such that a total of 6 LY3372689 dose levels were evaluated. Safety was assessed by adverse events (AE), safety laboratories, electrocardiograms, vital signs, physical exams, and neurological exams. Plasma PK was assessed up to 48 hours after LY3372689 administration.ResultA total of 23 HV (15 males, 8 females; 22 – 63 years) participated in the study, of which 18 completed and 5 did not complete due to loss to follow up procedures or did not meet randomization criteria. LY3372689 was generally well tolerated up to the maximum dose administered. There were no serious AE reported and no subjects discontinued the study because of an AE. A total of 13 treatment‐emergent AE reported by 5 subjects considered as possibly related to study treatment were mostly mild in severity, and included pain of skin, headache, limb discomfort, pain in extremity, chest discomfort, muscle fatigue, nausea, and rash macular. The PK of LY3372689 supported daily dosing and was generally dose proportional based on AUC and Cmax.ConclusionLY3372689 demonstrated an acceptable safety and PK profile following single oral doses of LY3372689 in HV, which supports further investigation of LY3372689 as a potential treatment for tauopathies.

Inhibition of BACE1 has become an important strategy in the quest for disease modifying agents to slow the progression of Alzheimer’s disease. We previously reported the fragment-based discovery of LY2811376, the first BACE1 inhibitor reported to demonstrate robust reduction of human CSF Aβ in a Phase I clinical trial. We also reported on the discovery of LY2886721, a potent BACE1 inhibitor that reached phase 2 clinical trials. Herein we describe the preparation and structure activity relationships (SAR) of a series of BACE1 inhibitors utilizing trans-cyclopropyl moieties as conformational constraints. The design, details of the stereochemically complex organic synthesis, and biological activity of these BACE1 inhibitors is described.

Here we report an optimized protocol for the asymmetric introduction of a fluorine atom into a quaternary center facilitated by d-proline, Selectfluor®, and trifluoroethanol. The synthesis proceeds over four steps starting from a chiral amino alcohol precursor and provides the desired enantiomer with no erosion of chiral purity and good diastereoselectivity. The process optimization allowed diastereoselective preparation of the key intermediate on a multigram scale.

Recent literature has indicated an amyloid-dependent increase in endogenous mouse Tau in the cerebrospinal fluid (CSF) of two APP-overexpressing transgenic mouse models. Long-term BACEI inhibitor treatment that blocks further plaque accrual has been shown to prevent additional CSF tau increases in APP transgenic mice with early and moderate Aβ pathology (J. Schelle et al., 2017, Alzheimer's & Dementia 13, 701-709). These findings led us to investigate the relationship between mouse CSF tau and Aβ pathology in the PDAPP transgenic mouse model. Additionally, we evaluated whether therapeutic removal of pre-existing plaque altered the elevated CSF mouse tau in PDAPP mice. ELISA and MSD immunoassays were used to measure brain Aβ 1-42 and CSF tau from PDAPP mice. PDAPP mice, aged 16-18 months, were treated for 4-16 weeks. Treatments included BACE inhibitors LY2811376 and LY2886721 in feed, mE8-IgG2a antibody, a combination of LY2811376 and mE8 antibody, control feed and IgG2a antibody. PDAPP mice showed a significant age-dependent increase in mouse tau from 3-23 months of age. Therapeutic intervention studies that led to plaque removal in aged PDAPP mice with the mE8 antibody had no effect on CSF tau, whereas LY2811376 treatment unexpectedly resulted in increased CSF tau. Since LY2811376 is known to inhibit Cathepsin D (Cat D), a follow-up study was performed with a BACE inhibitor, LY2886721, which avoids Cat D inhibition. We observed no change in the elevated CSF tau levels with 12 weeks of treatment of LY2886721, even though deposited plaque was lowered by approximately half. Endogenous mouse tau in PDAPP mouse CSF showed an age-dependent increase that likely is amyloid dependent. The removal of pre-existing plaque did not alter the elevated CSF tau levels in the aged PDAPP mouse model.

LY3202626 is a potent, freely CNS-penetrant small molecule BACE1 inhibitor in development for the treatment of Alzheimer’s disease (AD). The in vitro and nonclinical in vivo pharmacology of LY3202626 is described herein. LY3202626 was assessed for inhibition of recombinant hBACE1 using Fluorescence Resonance Energy Transfer (FRET)-based methodology. To determine effects on BACE1 activity in intact cells, LY3202626 was tested in primary cortical neurons prepared from Platelet Derived Amyloid Precursor Protein (PDAPP) transgenic mouse embryos. For determination of in vivoactivity, LY3202626 was characterized for acute effects on brain APP metabolism (i.e. sAPPβ, C99, and Aβ 1-x) in young female PDAPP mice. Finally, the pharmacokinetic/pharmacodynamic relationship of LY3202626-induced BACE1 inhibition in plasma and cerebrospinal fluid (CSF) was determined following acute oral dosing in beagle dogs. LY3202626 demonstrated potent hBACE1 and hBACE2 enzyme inhibition with IC50 values of 0.61 + 0.10 (n=5) and 0.87 + 0.24 (n=6) nM, respectively; assessment of LY3202626 activity against additional aspartyl proteases (e.g. cathepsin D, pepsin) resulted in IC50 values > 14,000 nM. LY3202626 produced a concentration dependent decrease of Aβ secretion in the PDAPP mouse primary 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 Aβ, sAPPβ, and C99 were observed in cortex and hippocampus of PDAPP mice. In beagle dog, acute oral dosing of 1.5 mg/kg LY3202626 resulted in lowering of plasma and CSF Aβ 1-x by approximately 80% at the nadir; the CSF Aβ 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 Aβ in both PDAPP mice and beagle dogs. 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.

LY3202626 is a small molecule non-selective BACE1 inhibitor. In dogs, LY3202626 is highly potent, producing dose-dependent reductions in CSF and plasma Aβ concentrations. The initial human study (I7X-EW-LLCA, clinicaltrials.gov identifier NCT02323334) described herein was a single and multiple ascending dose trial, in 4 parts, to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of LY3202626 in healthy subjects (HS) and patients with Alzheimer’s disease (AD). In Part A, HS received placebo or LY3202626 (0.1 to 45 mg) as single doses. Part B evaluated CSF PK/PD in HS by serial sampling after single dose placebo or LY3202626 (1.6 to 26 mg) administration. In Part C, HS received daily doses of placebo or LY3002626 (1 to 26 mg) for 14 days. In Part D, patients with AD received daily doses of 6 mg for 14 days. In Parts C and D, CSF was obtained by lumbar puncture at baseline and 24 hrs after last dose. Plasma and CSF LY3202626 concentrations were measured by LC/MS/MS. Plasma and CSF Aβ1-40 and Aβ1-42 were measured by immunoassay. PK/PD was assessed after single doses and at steady state. Adverse events, vital signs, ECG, clinical laboratories, neurological and eye exams were assessed throughout the study and for up to 42 days after the last dose to characterize safety and tolerability. LY3202626 was well tolerated by HS (n=92) and AD patients (n=2) at all doses. In HS, maximum plasma LY3202626 concentrations (Cmax) were observed approximately 3 hrs post-dose. The terminal half-life was around 21 hrs, and PK was generally dose proportional. LY3202626 appeared to freely penetrate the blood brain barrier, with CSF Cmax approximately 6 hrs post-dose. Plasma steady-state was achieved within 8 to 10 days. Following single doses, LY3202626 produced dose-dependent reductions in plasma and CSF Aβ1-40 and Aβ1-42. At steady-state, Aβ1-40 in CSF was reduced by approximately 50%, 75%, and >90% at daily doses of 1, 6, or 26 mg, respectively. LY3202626 is a well-tolerated, potent, low dose, freely CNS penetrant BACE inhibitor. These data support further clinical development of LY3202626.

LY3202626 is a potent, freely CNS-penetrant small molecule BACE1 inhibitor in development for the treatment of Alzheimer’s disease (AD). Herein, we demonstrate strong pharmacokinetic / pharmacodynamic (PK/PD) relationships in PDAPP mice between central LY3202626 exposure and central (hippocampal and cortical brain tissue) BACE1 inhibition as determined by quantifying markers of amyloid precursor protein (APP) metabolism. In a dose response study, young female PDAPP mice (n=6/group) were orally administered 0, 0.3, 1.0, or 3.0 mg LY3202626/kg and were sacrificed at 3 hr post-dose. In a separate time course study, young female PDAPP mice were sacrificed at 3, 6, 9, or 12 hours following a 3 mg LY3202626/kg oral dose. In all studies, LY3202626 concentrations were determined in plasma and brain samples by LC/MS/MS and concentrations of sAPPbeta, C99 and Abeta 1-X were determined in hippocampus and cortex using ELISA methodology. Oral administration of LY3202626 to PDAPP mice produced dose-dependent reductions in brain Abeta, C99, and sAPPbeta with LY3202626 brain concentrations negatively-correlated with all three PD endpoints (r2 values > 0.56). Changes in each BACE1 biomarker were similar in the cortical and hippocampal brain regions (all r2 values > 0.90). In a time-course study following a dose of 3 mg/kg LY3202626, free brain exposure over the 3-12 hour time-course study correlated well with hippocampal Abeta 1-X changes (r2 = 0.60), indicating that the robust PK/PD relationship was maintained over time. All correlations were significant, with p-values <0.0001. Observed free (unbound) concentrations in brain and plasma across these studies suggest LY3202626 is highly brain penetrant in PDAPP mice. LY3202626 is a potent inhibitor of BACE1. Administration of LY3202626 results in significant changes in central biomarkers of BACE1 activity, and the magnitude of these changes correlate well with observed.

The use of combination therapies is an emerging and exciting potential path forward for treatment of Alzheimer’s disease (AD). Our understanding of the AD cascade continues to evolve and show the proximal deposition of Aβ precedes cognitive impairment by 10 to 15 years and that deposition of Aβ has reached a plaque ceiling by the time mild to moderate symptoms manifest. Thus, an increasing belief in the field is that aggressive anti-amyloid interventions, such as combination therapy, may be necessary for treatment of symptomatic Alzheimer’s patients. In order to provide clinically translatable data that moves beyond simple proof-of-concept, we conducted a large dose response combination therapy study. Aged PDAPP mice (18-20 months, N = 285) were randomized into an eleven-arm study that consisted of a BACE inhibitor (LY2811376) monotherapy dose response, plaque-specific Aβ antibody mE8-IgG2a (anti-Aβp3-x) dose response plus high-dose BACE inhibitor, high-dose, plaque-specific Aβ antibody mE8-IgG2a (anti-Aβp3-x) plus BACE inhibitor dose response, and control/time zero cohorts. Animals were sacrificed after 4 months on study and samples were analyzed by various biochemical and immunohistological techniques. We observed a significant dose-response relationship on deposited Aβ1-42 (ELISA) for BACE inhibitor monotherapy compared to the control treated animals (-7%, -23%, and -60%). Additionally, histological analyses demonstrated BACE inhibitor treatment significantly lowered the diffuse deposits of Aβ. For combination drug treatment arms, we observed a significant broad dose response on deposited Aβ1-42 (ELISA) in the presence of high-dose antibody and varying dose levels of BACE inhibitor (from -33% through -84%). The combination drug treatments of a high-dose BACE inhibitor in the presence of varying dose levels of Aβ antibody revealed a significant, yet steeper dose response on deposited Aβ1-42 (from -60% through -84%). The histological analyses for deposited Aβ paralleled observed biochemical changes for combination therapy groups. Several of the of drug combination treatments resulted in significant synergistic lowering of deposited Aβ. The combination therapy showed a robust, highly significant and synergistic dose response relationship to plaque lowering in the transgenic mouse model. These results support rationale for using combination therapies in clinical trials.

Here, we report a continuous flow protocol for the [3 + 2] cycloaddition of nitrones, in situ generated from oximes, into bicyclic isoxazolidines. This thermal process required very high temperatures to be efficient that were not easily reached in conventional reactors. A couple of examples are presented and in both the flow process showed a greater performance than the batch mode. The process intensification study allowed the generation of 120 g/h of a key pharmaceutical intermediate.