This project will advance the re-positioning of a novel therapeutic agent, fasudil, for the treatment of Alzheimer?s disease (AD). Currently there is a huge unmet need for safe and effective disease modifying therapies to treat and prevent AD. This study will generate preclinical pharmacological outcomes to prepare for the USA FDA application for AD clinical trials and extend our studies of the mechanism of action (MOA) of fasudil in cells and the 3X-Tg AD mouse model of disease. Both PIs have extensive experience in the molecular biology and pathology of AD, the biological activity of fasudil and Wnt signaling, and in preclinical drug development; we are ideally positioned to evaluate the efficacy and the MOA of fasudil and test our hypothesis that fasudil down-regulates non-canonical (Planar Cell Polarity (PCP)) Wnt signaling and the downstream effects of Dickkopf-1 (Dkk1), leading to reduced Tau phosphorylation. Specifically, we will achieve three Aims.
Aim 1. To determine PK of fasudil in WT and 3X-Tg AD mice after acute and chronic oral dosing (PO). We will determine the brain/plasma ratio of novel fasudil metabolites after IV delivery of fasudil to mice, and then determine the time of maximum concentration observed (Tmax), half-life (t1/2), peak plasma concentration (Cmax), area under the curve (AUC), and bioavailability of fasudil after PO dosing. We will determine steady-state fasudil exposure in plasma and brain in a multiple-PO dosing paradigm, and finally confirm PK profile in 3X-Tg AD transgenic mice. We will specifically identify the most permeable fasudil metabolites and establish the active pharmaceutical ingredient for monitoring PK profiling. Data from acute dosing of fasudil will be used to model chronic dosing paradigm, and steady state fasudil levels in transgenic mouse brains under variable dosing paradigms will be established.
Aim 2. To determine PD of fasudil and establish PK-PD relationship in transgenic mice. We will quantify brain levels of A? (A?38, 40, 42), total Tau, and pTau in 3X-Tg AD transgenic mice after a single dose of fasudil as well as multiple doses of fasudil over a two-week interval. We will determine efficacy of chronic fasudil dosing on memory tests performed on transgenic mice and establish PK-PD relationship and calculate the therapeutic index.
Aim 3. To determine the mechanism of action for effect of fasudil on A?/Tau/pTau. We will examine the effect of fasudil on the Wnt-?-cat and Wnt-PCP pathways and phosphorylation of Tau. In addition, we will establish a causative relationship between regulation of Wnt pathway and reduction of A?/Tau/pTau. We will measure the dose- dependent impact of fasudil on ?-catenin, p-?-catenin, and promoter assays for both Wnt pathways. Results will be correlated with reduction of A?/Tau/pTau. We will also activate the Wnt-PCP pathway in HEK293 cells with Dkk1 and then treat with fasudil to determine the effect of fasudil on A?/Tau/pTau during PCP Wnt ac tivation.
This project will develop essential preclinical data on a new therapeutic agent, fasudil, for the treatment of Alzheimer?s disease (AD). Currently there is no cure for AD. There is a huge unmet need for safe and effective therapies to treat and prevent AD. Clinical trials of fasudil for AD have been approved in China and Japan, but not in the USA. This project will study the pharmacology and toxicity of fasudil to prepare for the USA FDA application for AD clinical trials. Amyloid ? protein (A?)-containing plaques and hyperphosphorylated Tau- containing neurofibrillary tangles are two hallmarks of AD. Our recent publication shows that fasudil efficiently removed ?-amyloid pathology in brains of a transgenic mouse model for AD. We hypothesize that fasudil blocks Planar Cell Polarity (PCP)) Wnt signaling and Dickkopf-1 (Dkk1), leading to reduced A? pathology and increased synaptic stability. Here we also propose to analyze the effects of fasudil on Tau-tangles.
