Scott A. Monk

Robust Central Reduction of Amyloid-β in Humans with an Orally Available, Non-Peptidic β-Secretase Inhibitor

According to the amyloid cascade hypothesis, cerebral deposition of amyloid-β peptide (Aβ) is critical for Alzheimers disease (AD) pathogenesis. Aβ generation is initiated when β-secretase (BACE1) cleaves the amyloid precursor protein. For more than a decade, BACE1 has been a prime target for designing drugs to prevent or treat AD. However, development of such agents has turned out to be extremely challenging, with major hurdles in cell penetration, oral bioavailability/metabolic clearance, and brain access. Using a fragment-based chemistry strategy, we have generated LY2811376 [(S)-4-(2,4-difluoro-5-pyrimidin-5-yl-phenyl)-4-methyl-5,6-dihydro-4H-[1,3]thiazin-2-ylamine], the first orally available non-peptidic BACE1 inhibitor that produces profound Aβ-lowering effects in animals. The biomarker changes obtained in preclinical animal models translate into man at doses of LY2811376 that were safe and well tolerated in healthy volunteers. Prominent and long-lasting Aβ reductions in lumbar CSF were measured after oral dosing of 30 or 90 mg of LY2811376. This represents the first translation of BACE1-driven biomarker changes in CNS from preclinical animal models to man. Because of toxicology findings identified in longer-term preclinical studies, this compound is no longer progressing in clinical development. However, BACE1 remains a viable target because the adverse effects reported here were recapitulated in LY2811376-treated BACE1 KO mice and thus are unrelated to BACE1 inhibition. The magnitude and duration of central Aβ reduction obtainable with BACE1 inhibition positions this protease as a tractable small-molecule target through which to test the amyloid hypothesis in man.

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.

COMBINATION THERAPY WITH A PLAQUE-SPECIFIC ABETA ANTIBODY AND A BACE INHIBITOR RESULTS IN DRAMATIC PLAQUE REDUCTION IN A DOSE-DEPENDENT MANNER IN AGED PDAPP TRANSGENIC MICE

lowed. Results:High antibody responses were found in both species. In the rabbit, 277.16 118.4 mg/ml plasmaweremeasured after 5 immunizations with no significant differences between the dose groups. Antibody levels declined slightly to 246.66 109.5 mg/ml after a resting period of two months. Low levels of IFNg and IL-17 secreting splenocytes were found in the ELISPOTassays from rabbit splenocytes. Rhesusmonkeys reachedmean antibody levels of 114.2 6 41.67 mg/ml plasma after five immunizations. The isotype profile was high on IgG4 antibodies, indicative of a Th2 immune response. After three immunizations, no IL-17 or IFNg producing cells were found in ELISPOT assays from PBMCs. Conclusions: DNA Ab42 immunization leads to high antibody titers in large mammals, and is likely to produce high antibody levels and a safe (Th2 biased) immune response in humans as well. supported by NIA/NIH P30AG12300-21, Zale Foundation, Rudman Foundation, AWARE, Presbyterian Village North, Freiberger, and Denker Family Funds

Simultaneous quantitation of the BACE1 inhibitor AZD3293 and its metabolite AZ13569724 in human matrices by LC–MS/MS

AIM AZD3293 is a novel BACE1 inhibitor in Phase III development for Alzheimers disease. Sensitive and robust bioanalytical methods were required to quantitate AZD3293 and its metabolite AZ13569724 in human biological matrices. METHODOLOGY/RESULTS Human plasma was prepared by protein precipitation. Linearity for both analytes was in the range of 0.5-500 ng/ml with up to 100-fold dilution. Plasma ultrafiltrate samples were prepared using Centrifree® ultrafiltration device. Urine and CSF samples were analyzed directly after dilution. A 27% decrease in AZD3293 concentrations in the CSF collection apparati was found due to nonspecific binding. Incurred sample reanalysis was acceptable. CONCLUSION Methods for simultaneous quantitation of AZD3293 and its metabolite AZ13569724 in human biological matrices have been validated and successfully applied to clinical studies.

Clinical Bioavailability of the Novel BACE1 Inhibitor Lanabecestat (AZD3293): Assessment of Tablet Formulations Versus an Oral Solution and the Impact of Gastric pH on Pharmacokinetics

The relative bioavailability of lanabecestat administered as 2 tablet formulations versus an oral solution was investigated. This phase 1 single‐center, open‐label, randomized, 3‐period crossover study involved healthy male and nonfertile female subjects aged 18–55 years (NCT02039180). Subjects received a single 50‐mg lanabecestat dose as solution, tablet A, or tablet B on day 1 of each crossover period; 14 of 16 subjects completed the study. Relative bioavailability based on plasma lanabecestat AUC0–∞ (area under the plasma drug concentration–time curve from zero to infinity) geometric mean ratio versus oral solution (primary variable) was: tablet A, 1.052 (90% confidence interval [CI], 1.001–1.106); tablet B, 1.040 (0.989–1.093). These 90%CIs for geometric mean ratios are within accepted standard bioequivalence boundaries for all other pharmacokinetic (PK) parameters for both lanabecestat and metabolite (AZ13569724). All 3 formulations had similar plasma lanabecestat concentration–time profiles. Six adverse events were reported by 6 subjects (37.5%, all mild). GastroPlus modeling predicted a negligible impact of gastric pH changes on systemic PK (up to pH 7.4). Both tablet formulations fall within standard accepted bioequivalence criteria versus the oral solution. A single 50‐mg lanabecestat dose was well tolerated as a solution or tablet formulation in this population.

LANABECESTAT DOES NOT AFFECT PHARMACOKINETICS OF THE BCRP SUBSTRATE ROSUVASTATIN

in the clinical outcomes. The decline in cognition and other clinical endpoints over 18 months in participants receiving verubecestat was similar across all exposure quartiles. The estimated slope of AUC effect on ADAS-cog disease progression was small (-4.4e-5) and the 90% CI contained zero. Conclusions:Overall, these results indicate that EPOCH tested drug exposures and target engagement consistent with 72% and 90% inhibition of Ab production at the 12 and 40 mg doses, respectively. Together with the lack of exposure-dependency in the cognition data, these results suggest that insufficient dose or target engagement was not the driver for the failure to meet the clinical endpoints as expected exposures were achieved. Ref 1. Clin Pharmacol Ther. 2015 Mar;97(3):210-4.

A CORRELATIONAL ANALYSIS OF EXPOSURE AND PHARMACODYNAMIC EFFECTS OF THE BACE1 INHIBITOR LY3202626 IN PDAPP MICE FOLLOWING ACUTE ORAL DOSING

Background: 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. Methods: In a dose response study, young female PDAPP mice (n1⁄46/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. Results: Oral administration of LY3202626 to PDAPPmice produced dose-dependent reductions in brain Abeta, C99, and sAPPbeta with LY3202626 brain concentrations negatively-correlated with all three PD endpoints (r values > 0.56). Changes in each BACE1 biomarker were similar in the cortical and hippocampal brain regions (all r values > 0.90). In a timecourse 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 (r 1⁄4 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. Conclusions: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.

A COMPARISON OF IN VIVO POTENCY IN TWO BACE INHIBITORS AS DETERMINED IN HUMANS AND DOGS

P1-365 A COMPARISON OF IN VIVO POTENCY IN TWO BACE INHIBITORS AS DETERMINED IN HUMANS AND DOGS Brian A. Willis, Patrick C. May, Scott A. Monk, Stephen L. Lowe, Stuart Friedrich, Dustin J. Mergott, Leonard Boggs, Hakop Gevorkyan, Stan Jhee, Robert A. Dean, Masako Nakano, Celedon R. Gonzales, Eli Lilly and Co., Indianapolis, Indiana, United States; Eli Lilly and Company, Indianapolis, Indiana, United States; Lilly NUS Center of Clinical Pharmacology, Singapore, Singapore; Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Co., Indianapolis, Indiana, United States; Parexel International, Glendale, California, United States; Eli Lilly Japan K.K., Kobe, Japan. Contact e-mail: p.c. may@lilly.com