Charles E. Nolan

Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimers disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Aβ (β-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

Concentration-Dependent Modulation of Amyloid-β in Vivo and in Vitro Using the γ-Secretase Inhibitor, LY-450139

LY-450139 is a γ-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-β (Aβ) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Aβ responses to LY-450139 in the guinea pig, a nontransgenic model that has an Aβ sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2–60 mg/kg), and brain, cerebrospinal fluid, and plasma Aβ levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Aβ levels at early time points, with return to baseline within hours. Higher doses inhibited Aβ levels in all compartments at early time points, but elevated plasma Aβ levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3–30 mg/kg/day) for 5 days. Plasma Aβ was significantly inhibited at 10–30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Aβ elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Aβ were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the γ-secretase complex, eliciting Aβ lowering at high concentrations but Aβ elevation at low concentrations.

Metabolism-Directed Design of Oxetane-Containing Arylsulfonamide Derivatives as γ-Secretase Inhibitors

A metabolism-based approach toward the optimization of a series of N-arylsulfonamide-based γ-secretase inhibitors is reported. The lead cyclohexyl analogue 6 suffered from extensive oxidation on the cycloalkyl motif by cytochrome P450 3A4, translating into poor human liver microsomal stability. Knowledge of the metabolic pathways of 6 triggered a structure-activity relationship study aimed at lowering lipophilicity through the introduction of polarity. This effort led to several tetrahydropyran and tetrahydrofuran analogues, wherein the 3- and 4-substituted variants exhibited greater microsomal stability relative to their 2-substituted counterparts. Further reduction in lipophilicity led to the potent γ-secretase inhibitor and 3-substituted oxetane 1 with a reduced propensity toward oxidative metabolism, relative to its 2-substituted isomer. The slower rates of metabolism with 3-substituted cyclic ethers most likely originate from reductions in lipophilicity and/or unfavorable CYP active site interactions with the heteroatom. Preliminary animal pharmacology studies with a representative oxetane indicate that the series is generally capable of lowering Aβ in vivo. As such, the study also illustrates the improvement in druglikeness of molecules through the use of the oxetane motif.

Spirocyclic Sulfamides as β-Secretase 1 (BACE-1) Inhibitors for the Treatment of Alzheimer’s Disease: Utilization of Structure Based Drug Design, WaterMap, and CNS Penetration Studies To Identify Centrally Efficacious Inhibitors

β-Secretase 1 (BACE-1) is an attractive therapeutic target for the treatment and prevention of Alzheimers disease (AD). Herein, we describe the discovery of a novel class of BACE-1 inhibitors represented by sulfamide 14g, using a medicinal chemistry strategy to optimize central nervous system (CNS) penetration by minimizing hydrogen bond donors (HBDs) and reducing P-glycoprotein (P-gp) mediated efflux. We have also taken advantage of the combination of structure based drug design (SBDD) to guide the optimization of the sulfamide analogues and the in silico tool WaterMap to explain the observed SAR. Compound 14g is a potent inhibitor of BACE-1 with excellent permeability and a moderate P-gp liability. Administration of 14g to mice produced a significant, dose-dependent reduction in central Aβ(X-40) levels at a free drug exposure equivalent to the whole cell IC(50) (100 nM). Furthermore, studies of the P-gp knockout mouse provided evidence that efflux transporters affected the amount of Aβ lowering versus that observed in wild-type (WT) mouse at an equivalent dose.

Cerebrospinal Fluid Amyloid-β (Aβ) as an Effect Biomarker for Brain Aβ Lowering Verified by Quantitative Preclinical Analyses

Reducing the generation of amyloid-β (Aβ) in the brain via inhibition of β-secretase or inhibition/modulation of γ-secretase has been pursued as a potential disease-modifying treatment for Alzheimers disease. For the discovery and development of β-secretase inhibitors (BACEi), γ-secretase inhibitors (GSI), and γ-secretase modulators (GSM), Aβ in cerebrospinal fluid (CSF) has been presumed to be an effect biomarker for Aβ lowering in the brain. However, this presumption is challenged by the lack of quantitative understanding of the relationship between brain and CSF Aβ lowering. In this study, we strived to elucidate how the intrinsic pharmacokinetic (PK)/pharmacodynamic (PD) relationship for CSF Aβ lowering is related to that for brain Aβ through quantitative modeling of preclinical data for numerous BACEi, GSI, and GSM across multiple species. Our results indicate that the intrinsic PK/PD relationship in CSF is predictive of that in brain, at least in the postulated pharmacologically relevant range, with excellent consistency across mechanisms and species. As such, the validity of CSF Aβ as an effect biomarker for brain Aβ lowering is confirmed preclinically. Meanwhile, we have been able to reproduce the dose-dependent separation between brain and CSF effect profiles using simulations. We further discuss the implications of our findings to drug discovery and development with regard to preclinical PK/PD characterization and clinical prediction of Aβ lowering in the brain.

Pharmacodynamics and Pharmacokinetics of the γ-Secretase Inhibitor PF-3084014

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Aβ. To further characterize Aβ dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Utilizing Structures of CYP2D6 and BACE1 Complexes To Reduce Risk of Drug-Drug Interactions with a Novel Series of Centrally Efficacious BACE1 Inhibitors.

In recent years, the first generation of β-secretase (BACE1) inhibitors advanced into clinical development for the treatment of Alzheimer’s disease (AD). However, the alignment of drug-like properties and selectivity remains a major challenge. Herein, we describe the discovery of a novel class of potent, low clearance, CNS penetrant BACE1 inhibitors represented by thioamidine 5. Further profiling suggested that a high fraction of the metabolism (>95%) was due to CYP2D6, increasing the potential risk for victim-based drug–drug interactions (DDI) and variable exposure in the clinic due to the polymorphic nature of this enzyme. To guide future design, we solved crystal structures of CYP2D6 complexes with substrate 5 and its corresponding metabolic product pyrazole 6, which provided insight into the binding mode and movements between substrate/inhibitor complexes. Guided by the BACE1 and CYP2D6 crystal structures, we designed and synthesized analogues with reduced risk for DDI, central efficacy, and improved hERG therapeutic margins.

Discovery of a Series of Efficient, Centrally Efficacious BACE1 Inhibitors through Structure-Based Drug Design.

The identification of centrally efficacious β-secretase (BACE1) inhibitors for the treatment of Alzheimers disease (AD) has historically been thwarted by an inability to maintain alignment of potency, brain availability, and desired absorption, distribution, metabolism, and excretion (ADME) properties. In this paper, we describe a series of truncated, fused thioamidines that are efficiently selective in garnering BACE1 activity without simultaneously inhibiting the closely related cathepsin D or negatively impacting brain penetration and ADME alignment, as exemplified by 36. Upon oral administration, these inhibitors exhibit robust brain availability and are efficacious in lowering central Amyloid β (Aβ) levels in mouse and dog. In addition, chronic treatment in aged PS1/APP mice effects a decrease in the number and size of Aβ-derived plaques. Most importantly, evaluation of 36 in a 2-week exploratory toxicology study revealed no accumulation of autofluorescent material in retinal pigment epithelium or histology findings in the eye, issues observed with earlier BACE1 inhibitors.

Quantitative Pharmacokinetic/Pharmacodynamic Analyses Suggest That the 129/SVE Mouse Is a Suitable Preclinical Pharmacology Model for Identifying Small-Molecule γ-Secretase Inhibitors

Alzheimers disease (AD) poses a serious public health threat to the United States. Disease-modifying drugs slowing AD progression are in urgent need, but they are still unavailable. According to the amyloid cascade hypothesis, inhibition of β- or γ-secretase, key enzymes for the production of amyloid β (Aβ), may be viable mechanisms for the treatment of AD. For the discovery of γ-secretase inhibitors (GSIs), the APP-overexpressing Tg2576 mouse has been the preclinical model of choice, in part because of the ease of detection of Aβ species in its brain, plasma, and cerebrospinal fluid (CSF). Some biological observations and practical considerations, however, argue against the use of the Tg2576 mouse. We reasoned that an animal model would be suitable for GSI discovery if the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a compound for Aβ lowering in this model is predictive of that in human. In this study, we assessed whether the background 129/SVE strain is a suitable preclinical pharmacology model for identifying new GSIs by evaluating the translatability of the intrinsic PK/PD relationships for brain and CSF Aβ across the Tg2576 and 129/SVE mouse and human. Using semimechanistically based PK/PD modeling, our analyses indicated that the intrinsic PK/PD relationship for brain Aβx-42 and CSF Aβx-40 in the 129/SVE mouse is indicative of that for human CSF Aβ. This result, in conjunction with practical considerations, strongly suggests that the 129/SVE mouse is a suitable model for GSI discovery. Concurrently, the necessity and utilities of PK/PD modeling for rational interpretation of Aβ data are established.

Pharmacodynamics and pharmacokinetics of the gamma-secretase inhibitor PF-3084014.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Aβ. To further characterize Aβ dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.