Robust Pharmacodynamic Effect of LY3202626, a Central Nervous System Penetrant, Low Dose BACE1 Inhibitor, in Humans and Nonclinical Species.

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-ß (Aß) levels was evaluated in a series of in vitro assays, as well as in in vivo and multi-part clinical pharmacology studies. Aß 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 Aß1-40 and 0.228±0.244 nM for Aß1-42 in PDAPP neuronal cultures. In dogs, CSF Aß1hboxx concentrations were significantly reduced by ∼80% at 9 hours following a 1.5 mg/kg dose. In humans, CSF Aß1-42 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.

Discovery and Early Clinical Development of LY3202626, a Low-Dose, CNS-Penetrant BACE Inhibitor.

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.

Excretion, Mass Balance, and Metabolism of [14C]LY3202626 in Humans: An Interplay of Microbial Reduction, Reabsorption, and Aldehyde Oxidase Oxidation That Leads to an Extended Excretion Profile.

The mass balance, excretion, and metabolism of LY3202626 were determined in healthy subjects after oral administration of a single dose of 10 mg of (approximately 100 µCi) [14C]LY3202626. Excretion of radioactivity was slow and incomplete, with approximately 75% of the dose recovered after 504 hours of sample collection. The mean total recovery of the radioactive dose was 31% and 44% in the feces and urine, respectively. Because of low plasma total radioactivity, plasma metabolite profiling was conducted by accelerator mass spectrometry. Metabolism of LY3202626 occurred primarily via O-demethylation (M2) and amide hydrolysis (M1, M3, M4, and M5). Overall, parent drug, M1, M2, and M4 were the largest circulating components in plasma, and M2 and M4 were the predominant excretory metabolites. The slow elimination of total radioactivity was proposed to result from an unusual enterohepatic recirculation pathway involving microbial reduction of metabolite M2 to M16 in the gut and reabsorption of M16, followed by hepatic oxidation of M16 back to M2. Supporting in vitro experiments showed that M2 is reduced to M16 anaerobically in fecal homogenate and that M16 is oxidized in the liver by aldehyde oxidase to M2. LY3202626 also showed a potential to form a reactive sulfenic acid intermediate. A portion of plasma radioactivity was unextractable and presumably bound covalently to plasma proteins. In vitro incubation of LY3202626 in human liver microsomes in the presence of NADPH with dimedone as a trapping agent implicated the formation of the proposed sulfenic acid intermediate. SIGNIFICANCE STATEMENT: The excretion of radioactivity in humans after oral administration of a single dose of 10 mg of [14C]LY3202626 was very slow. The results from in vitro experiments suggested that an interplay between microbial reduction, reabsorption, and aldehyde oxidase oxidation (M2 → M16 → M2) could be a reason for extended radioactivity excretion profile. In vitro metabolism also showed that LY3202626 has the potential to form a reactive sulfenic acid intermediate that could potentially covalently bind to plasma protein and result in the observed unextractable radioactivity from plasma.

Assessment of Transporter Polymorphisms as a Factor in a BCRP Drug Interaction Study With Lanabecestat.

Lanabecestat is a human ß-site amyloid precursor protein-cleaving enzyme 1 inhibitor in development to slow disease progression in patients with early Alzheimer's disease. The study evaluated the breast cancer resistance protein (BCRP) inhibition potential of lanabecestat on the pharmacokinetics (PK) of rosuvastatin, a probe for BCRP activity, in healthy white subjects who were not carriers of SLCO1B1 (c.521T>C), not homozygotes for ABCG2 (c.421C>A or c.34G>A), and not heterozygotes of ABCG2 (c.421C>A and c.34G>A). The safety of lanabecestat + rosuvastatin, the effects of rosuvastatin on the PK of lanabecestat, and the effects of multiple genetic polymorphisms on rosuvastatin exposure were assessed. Geometric mean ratios of the maximum observed rosuvastatin concentration (Cmax ), area under the rosuvastatin concentration-versus-time curve (AUC) from time 0 to infinity, and time of maximum observed drug concentration (tmax ) when rosuvastatin was administered alone and with lanabecestat were contained within 0.8-1.25, as were lanabecestat AUC at steady state and tmax at steady state when lanabecestat was administered alone or with rosuvastatin. Lanabecestat Cmax at steady state increased 8% in the presence of rosuvastatin. Except for an approximately 80% increase of rosuvastatin AUC (P < .05) in the heterozygotes of ABCG2 c.421C>A relative to the CC genotype, there were no statistically significant associations between rosuvastatin exposure and polymorphisms assessed. Lanabecestat + rosuvastatin was associated with few treatment-emergent adverse events, all of which resolved and were mild. Lanabecestat does not meaningfully impact BCRP activity; therefore, restriction of concomitant administration with BCRP substrates, such as rosuvastatin, may be unnecessary.

Clinically Negligible Pharmacokinetic and Pharmacodynamic Interactions Between Lanabecestat and Dabigatran Etexilate, a Prototypical P-gp Substrate.

Lanabecestat, a novel ß-site amyloid precursor protein-cleaving enzyme 1 inhibitor evaluated for Alzheimer treatment, inhibits P-glycoprotein (P-gp) activity in vitro. After oral 50-mg lanabecestat administration, gastric fluid lanabecestat concentrations exceed half-maximal inhibitory concentration (IC50 ), suggesting P-gp inhibition at the intestinal wall. Plasma drug concentrations following 50 mg lanabecestat administered once daily are <10% of IC50 , suggesting minimal systemic P-gp interaction. Dabigatran etexilate (DE) is the prodrug of dabigatran, a thrombin inhibitor and P-gp substrate, making dabigatran exposure an intestinal P-gp activity indicator. This study (NCT02568397) was conducted in 60 healthy subjects receiving a single dose of 150 mg DE alone or during a lanabecestat treatment regimen. On day 16, lanabecestat and DE were coadministered; on day 20, DE was dosed 4 hours after lanabecestat. Safety was assessed using clinical labs, electrocardiogram, vital signs, Columbia Suicide Severity Rating Scale scores, adverse events, and eye and skin examinations. Pharmacokinetic/pharmacodynamic samples were collected up to 36 hours postdose. Geometric mean plasma dabigatran area under the curve from time 0 to infinity (AUC0-∞ ) and the maximum plasma drug concentration (Cmax ) increased by 15% and 17%, respectively, when coadministered with lanabecestat. When DE was dosed 4 hours after lanabecestat, there was no effect on plasma dabigatran AUC0-∞ , Cmax , or thrombin time. DE had no effect on lanabecestat's AUC0-∞ and Cmax at steady state (day 16) versus lanabecestat alone (day 15). No clinically relevant safety concerns were observed. Lanabecestat has no clinically meaningful effect on dabigatran exposure or on P-gp activity at the intestinal wall.

Preparation and biological evaluation of BACE1 inhibitors: Leveraging trans-cyclopropyl moieties as ligand efficient conformational constraints.

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 Aß 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.

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.

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 Alzheimer's 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.

A Model for Single Neuron Activity With Refractory Effects and Spike Rate Estimation Techniques.

The use of random point processes as models for neural spike trains allows the derivation of powerful statistical estimation techniques for time varying firing rates. Frequently, however, such estimators are based on the assumption that spike sequences follow a Poisson point process. Because of the bio-physical properties of neuronal action potentials, spike trains are affected by the refractory phenomenon that induces history dependency, and hence contradicts the Poisson assumption. In this work we present a neural spiking model, and a Maximum Likelihood (ML) estimation framework for time varying firing rates, that account for history dependencies in spike trains. Our framework is based on an exponential of polynomial model for the excitation function (stimulus), that generates a self exciting point process representing spike trains with absolute as well as relative refractory effects. Using this framework we employ techniques based on non-convex optimization and model order selection to derive ML estimators for neuronal firing rates. Results on simulated data with a refractory period show an improvement in accuracy when our estimation technique, that accounts for the complete refractory phenomenon, is used. Employing this estimation method for measured neuronal data shows an improvement in goodness of fit over estimators that do not account for the refractory effect, and also over other commonly used techniques.

Preparation and biological evaluation of conformationally constrained BACE1 inhibitors.

The BACE1 enzyme is a key target for Alzheimer's disease. During our BACE1 research efforts, fragment screening revealed that bicyclic thiazine 3 had low millimolar activity against BACE1. Analysis of the co-crystal structure of 3 suggested that potency could be increased through extension toward the S3 pocket and through conformational constraint of the thiazine core. Pursuit of S3-binding groups produced low micromolar inhibitor 6, which informed the S3-design for constrained analogs 7 and 8, themselves prepared via independent, multi-step synthetic routes. Biological characterization of BACE inhibitors 6-8 is described.

The potent BACE1 inhibitor LY2886721 elicits robust central Aß pharmacodynamic responses in mice, dogs, and humans.

BACE1 is a key protease controlling the formation of amyloid ß, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimer's 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 ß lowering in nonclinical animal models. Similar potent and persistent amyloid ß 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.

Robust central reduction of amyloid-ß in humans with an orally available, non-peptidic ß-secretase inhibitor.

According to the amyloid cascade hypothesis, cerebral deposition of amyloid-ß peptide (Aß) is critical for Alzheimer's disease (AD) pathogenesis. Aß generation is initiated when ß-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 Aß-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 Aß 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 Aß reduction obtainable with BACE1 inhibition positions this protease as a tractable small-molecule target through which to test the amyloid hypothesis in man.

Reversible stepwise negative regulation of CYP1A1 in cultured rat epidermal cells.

When serially passaged, rat epidermal keratinocytes lose the inducibility of CYP1A1 gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. In present experiments, loss of CYP1A1 inducibility occurred in a stepwise fashion, with some keratinocyte lines progressing through a transiently inducible state before becoming completely uninducible. The negative regulation occurred at the level of transcription, but the aryl hydrocarbon receptor (AhR) pathway appeared fully functional. Transient and stable transfection of uninducible cells with reporter constructs containing up to 4.2kb of the CYP1A1 5'-flanking region resulted in a TCDD-inducible increase in luciferase activity, despite no induction of the endogenous gene. Co-treatment with protein synthesis inhibitors and TCDD restored responsiveness of the endogenous CYP1A1 gene, indicating that the negative regulation was reversible and likely mediated by a labile protein. Together, these results demonstrate a novel mechanism of CYP1A1 transcriptional repression that does not involve any previously reported negative regulatory elements for CYP1A1.