Volumetric MRI and MRS provide sensitive measures of Alzheimer's disease neuropathology in inducible Tau transgenic mice (rTg4510).

The purpose of this study was to determine if in vivo high resolution 3D MRI and localized (1)H MR spectroscopy (MRS) can detect brain findings resembling Alzheimer's disease in a transgenic mouse model of Tau pathology. Seven double transgenic rTg4510 female mice and 7 age-matched wild-type (wt) female mice were evaluated at 5 months of age. To confirm the usefulness and consistency of in vivo MRI/S, we also scanned the brains of 14 male mice (7 rTg4510 and 7 age-matched wt) at 8 months of age. Mean hippocampal and cerebral cortex volumes in the female rTg4510 mice were 26.7% and 20.6% smaller than that in the wt controls (p<0.0001), respectively. Mean hippocampal and cerebral cortex volumes in the male rTg4510 mice were 18.4% and 16.9% smaller than that in the wt controls (p<0.00005), respectively. The mean volumes of the cerebellum were not statistically different between the rTg4510 and the wt groups. MRS assessment revealed that the myo-inositol to total creatine ratios (mIns/tCr), a measure of gliosis, were significantly higher in the hippocampus of rTg4510 mice relative to wt mice (p=0.03 for the females; p=0.005 for the males). Immunohistochemistry and histology in the same animals verified previously published data showing elevation of hyperphosphorylated Tau, glial activation and cortical and hippocampal neuronal loss. This study demonstrates that in vivo MRI/S can be a non-invasive biomarker to assess brain atrophy and related biochemical changes in the rTg4510 mouse model.

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 ß-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aß in guinea pigs. The therapeutic index between Aß reductions and changes in B-cell populations were studied for compound 10 h.

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 Aß 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 Aß EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

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 gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) 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 EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta 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 Abeta. To further characterize Abeta 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 Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta 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 Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Peripheral elevation of IGF-1 fails to alter Abeta clearance in multiple in vivo models.

Increasing beta-amyloid (Abeta) clearance may alter the course of Alzheimer's disease progression and attenuate amyloid plaque pathology. Insulin-like growth factor I (IGF-1) augmentation has been suggested to increase Abeta clearance by facilitating transport of Abeta out of the brain. The availability of safe agents that increase IGF-1 levels therefore makes IGF-1 elevation an attractive target for disease modifying therapy in AD. The present series of studies sought to replicate published paradigms in which peripheral IGF-1 administration lowered brain Abeta acutely, with reduction in plaque pathology after chronic treatment. Thus Abeta levels were measured in several animal models following treatments that elevated IGF-1. Administration of IGF-1 to young or old rats for up to 3 days had no effect on Abeta levels in brain, CSF, or plasma. In adult beagles, 4 days of dosing with the growth hormone secretagogue, CP-424391, doubled baseline plasma IGF-1 levels, yet failed to alter CSF or plasma Abeta. 5-day treatment of young Tg2576 mice with IGF-1 produced robust elevations of IGF-1 levels in plasma, but no effects on Abeta were detected in brain, CSF, or plasma. Finally, 11-month-old Tg2576 mice were implanted with subcutaneous minipumps delivering IGF-1 for 1 month. No significant changes in Abeta (by ELISA or Western blot), plaque pathology, or phospho-tau epitopes were detected. These results do not demonstrate acute or chronic actions of peripherally administered IGF-1 on Abeta levels or the phosphorylation state of tau and therefore do not suggest any disease-modifying benefits of IGF-1 restorative therapy for AD through these mechanisms.

Concentration-dependent modulation of amyloid-beta in vivo and in vitro using the gamma-secretase inhibitor, LY-450139.

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta 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 Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta 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 Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta 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 Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.