Characterization of Hit Compounds Identified from High-throughput Screening for their Effect on Blood–brain Barrier Integrity and Amyloid-β Clearance: In Vitro and In Vivo Studies

Abstract

In Alzheimer's disease (AD) the blood-brain barrier (BBB) is compromised, thus therapeutic targeting of the BBB to enhance its integrity and function could be a unique approach to treat, slow or hold the progression of AD. Recently, we have developed an in vitro high-throughput screening assay to screen for compounds that increase the integrity of a cell-based BBB model. Results from primary screen identified multiple hit compounds that enhanced the monolayer integrity. Herein, further characterization of selected hit compounds, namely 8-bromoguanosine cyclic monophosphate, JW74, 1,10-phenanthroline monohydrate, SB216763 and α-tocopherol was performed. Compounds were subjected to concentration-dependent studies to determine their EC50 and potency to enhance the cell-based model integrity by the Lucifer Yellow permeability and amyloid-beta (Aβ) transport across the monolayer. The compounds demonstrated different EC50s to enhance the monolayer integrity ranging from 0.4 to 12.8 µM, and different effect on enhancing Aβ transport with highest transport observed for α-tocopherol (2.2-fold increase). Such effects were associated with increased levels of tight junction proteins such as claudin-5 and/or ZO-1, and Aβ major transport proteins LRP1 and P-glycoprotein. In vivo studies for α-tocopherol were performed in AD mouse model; consistent with the in vitro results α-tocopherol significantly increased BBB integrity measured by IgG extravasation, and reduced brain Aβ levels. In conclusion, findings support our developed cell-based BBB model as a functional predictive in vivo tool to select hit compounds, and suggest that enhancing BBB tightness and function has the potential to reduce Aβ pathology associated with AD.

Multi-faceted therapeutic strategy for treatment of Alzheimer's disease by concurrent administration of etodolac and α-tocopherol

“…In addition, recent findings from our lab demonstrated etodolac to enhance the monolayer integrity in the absence and presence of Aβ oligomers (Qosa, Mohamed, Al Rihani et al, 2016); thus in this work, this experiment was performed in the absence of the stressor. Consistent with our previously published HTS studies (Qosa, Mohamed, Al Rihani et al, 2016;Elfakhri et al, 2018), etodolac and α-tocopherol each significantly enhanced tightness of the in vitro cell-based BBB model in a concentration dependent manner as demonstrated by reduction of the paracellular permeability marker 14 C-inulin across the bEnd3 monolayer when compared to the control (Fig. 1a).…”

“…To determine the effect of etodolac, α-tocopherol, and combination on Aβ transport across the monolayer, transport study of 125 I-Aβ 40 (TQ B→A ) was performed as reported previously (Qosa, Abuasal et al 2014;Elfakhri et al, 2018). Each of the three treatments increased the transport of 125 I-Aβ 40 in a concentration-dependent manner (Fig.…”

“…As each cage housed 2-3 mice, one jar containing 20 g Aqua-Jel (containing 1.25 mg α-tocopherol) was placed in each cage. While α-tocopherol administration by its addition to Aqua-Jel could introduce variability in the administered dose, a concern which is similar to that of adding drugs to food or drinking water, these methods have been used and showed acceptable results (Batarseh et al, 2018;Evangelista et al, 2018;Elfakhri et al, 2018;Qosa et al, 2012). The combination group received 10 mg/kg/day etodolac (IP) and supplemented with 10 mg/kg/day of α-tocopherol mixed with Aqua-Jel for one month.…”

“…Brain microvessels were isolated as described previously (Elfakhri et al, 2018). To brain homogenate, one volume of 30% Ficoll 400 (Sigma-Aldrich) was added.…”

“…To this end, we previously developed an in vitro high-throughput screening (HTS) assay to evaluate compounds based on their ability to improve barrier integrity and function in a cellbased BBB model (Qosa, Mohamed, Al Rihani et al, 2016;Elfakhri et al, 2018). Screening results indicated etodolac and α-tocopherol as strong candidate drugs for further evaluation.…”