James E. Finley

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.

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.

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.

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: Discovery of PF-3084014

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

Pharmacodynamics and Pharmacokinetics of the -Secretase Inhibitor PF-3084014 □ S

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.

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10 h.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

P4-254: Microglia catalyze the “refolding” of Aβ peptide into amyloid, suggesting an alternate mechanism for neuritic plaque formation

ease (HD), 2 Dementia lacking distinctive histology (DLDH), 2 Alzheimer’s disease (AD), 1 Atypical Tau and 1 Neuronal Intermediate Filament Inclusion Disease (NIFID)). Results: Urea fractions demonstrated cleaved 25kD TDP-43 fragments and HMWS in 20 cases including 18 FTLD-U (8 FTLD-U1, 5 FTLD-U2, 5 FTLD-U3), 1 MND and 1 DLDH. Three cases (1 FTLD-U3, 1 CBD and 1 MND) showed 25kD/45kD fragments with no associated HMWS and 16 cases (5 FTLD-U (2 FTLD-U1, 2 FTLD-U2 and 1 FTLD3), 5 Pick’s-type histology, 1 FTDP-17T, 1 CBD, 1 AD, 1 Huntington’s disease, 1 NIFID and 1 PSP) showed a HMWS with no associated 25kD fragments. A prominent 15-17kD fragment was also detected within sarkosyl soluble fractions in 41 cases, however, this was not significantly associated with any pathological type. Conclusions: These data indicate that TDP-43 processing and deposition is highly heterogeneous and the biochemical profile of TDP-43 may be more complex than previously described.