Grantee Research Project Results
2021 Progress Report: Integrated blood brain barrier – computational model development to predict doses ofconcern for compound linked neurotoxicity
EPA Grant Number: R840027Title: Integrated blood brain barrier – computational model development to predict doses ofconcern for compound linked neurotoxicity
Investigators: Knipp, Gregory , Stratford, Robert , Sluka, James
Current Investigators: Knipp, Gregory , Sluka, James
Institution: Purdue University , Indiana University
EPA Project Officer: Spatz, Kyle
Project Period: August 1, 2020 through July 30, 2023
Project Period Covered by this Report: August 1, 2020 through July 31,2021
Project Amount: $790,441
RFA: Advancing Toxicokinetics for Efficient and Robust Chemical Evaluations (2019) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
Objective 1: Adapt a presently available low-throughput triculture model of the human BBB to a higher throughput screen that will measure bi-directional compound permeability.
We outlined a goal to validate the human layered blood brain barrier triculture (human astrocytes, pericytes, and the HBEC-5i human brain microvessel endothelial cell line) model to assess the permeability of 50 established neurotoxicants. We completed validation of the BBB triculture model demonstrating significant barrier properties and could discriminate the permeation rates of marker compounds. The rank order of the permeability coefficients for these compounds matched their observed in vivo exposure and completed the data for our first project publication [K.E. Lubin and G.T. Knipp. Design of Experiment Based Optimization of an In Vitro Direct Contact Triculture Blood Brain Barrier Model for Permeability Screening. Pharm. Pharmacol. Int. J. 9(4):143-158 (2021)]. We began to follow up early studies where a former student added SH-SY5Y neurons to the basal chamber in an attempt to study permeability-linked neurotoxicity. Here we acquired primary midbrain neurons and incubated the BBB triculture with the neurons for three days prior to determining the permeability rates of marker compounds. Unfortunately, the primary neurons did not grow at a sufficient rate and it was clear that we would need more cells and significant method development to establish the model. Since we proposed to work primarily on bidirectional permeability across the BBB triculture model, we have set aside this work for now. As discussed below, another factor was the inability to acquire additional primary neurons from vendors.
We are conducting bidirectional permeability studies across the blood-brain barrier (BBB) triculture model with the selected 50 neurotoxicants. The permeability measurements for the following representative compounds have been performed with most being analyzed by HPLC include chlorpyrifos, bisphenol A, Di-(2-ethylhexyl) phthalate, thimerosal, acrylamide, belinostat, cisplatin, methoxychlor, nicotine, prazosin, caffeine, and perchlorate. We have measured permeability coefficients for a novel antiviral hit compound (DP3C01) that has high potency for Ebolavirus, but was neurotoxic in neuronal screen. Selected apparent permeability coefficients are reported in Table 1. We have found that a few of these compounds need to be repeated due to the fact that sink conditions (>90% of the compound in the donor chamber) was not maintained at the first measurement time point. We are repeating these compounds with sampling being adjusted to every five minutes. While these observations hinder the ability to calculate an apparent permeability coefficient, it does indicate that cisplatin, methoxychlor, and perchlorate will readily permeate the BBB and demonstrates that there exists a high probability of a neurotoxic event if these compounds are present in the bloodstream at higher concentrations. We are running additional permeability studies and are acquiring more neurotoxicants to continue throughout the upcoming year with the goal to complete all bidirectional permeability measurements by December of 2022. This will enable us to complete Objective 1 of adapting a presently available low-throughput triculture model of the human BBB.
Table 1. Representative apparent permeability coefficients (Papp) and the standard deviations are reported for the selected neurotoxicant and control compounds
Compound | Apical to Basolateral Papp (cm/sec) | Basolateral to Apical Papp (cm/sec) | Efflux Ratio (B-A Papp / A-B Papp) |
Nicotine | 3.10 x 10-7 ± 2.78 x 10-8 | 4.96 x 10-7 ± 1.65 x 10-7 | 1.6 |
Bisphenol A | 4.62 x 10-6 ± 2.24 x 10-6 | > 5.0 x 10-5 | > 10.8* |
Thimerosal | 2.82 x 10-5 ± 4.83 x 10-6 | 1.99 X 10-5 ± 2.22 x 10-6 | 0.71 |
Belinostat | 3.11 x 10-5 ± 8.85 x 10-6 | 2.89 X 10-7 ± 1.3 x 10-8 | 0.0093 |
DP3C01 | 4.61 x 10-6 ± 8.85 x 10-6 | 1.06 x 10-5 ± 4.32 x 10-7 | 2.29 |
Methoxychlor | > 5.0 x 10-5 | > 5.0 x 10-5 | |
Perchlorate | > 5.0 x 10-5 | > 5.0 x 10-5 |
We encountered significant equipment issues primarily arising from the current SARS-CoV2 pandemic, leading to delays in experimental progress. Our HPLC autosampler had a broken part that could not be replaced. A refurbished Agilent 1100 autosampler was purchased for the laboratory and upon installation and issues with communication between the instrument and the data acquisition software were ultimately resolved with online support. Another refurbished Agilent 1100 HPLC system has been purchased along with a second software license for data acquisition. These purchases were not made with EPA funding.
Significant delays in acquiring laboratory materials, such as reduced availability of essential consumables, reagents, and cells, were due to confounding effects of the SARS-CoV2 pandemic. One example is the availability of a growth factor media supplement (endothelial cell growth supplement; ECGS) supplied through Sigma-Aldrich. Briefly, ECGS acquisition was delayed due to a company shutdown of the German supplier. An initial one-month delay, led to several additional months. A separate source (Lifeline) for the ECGS was identified and used, followed by a head-to-head comparison of the two sources. The results demonstrated that the sources where interchangeable and we were able to bridge the materials gap until receiving the original Sigma Aldrich ECGS order in May 2021. During Spring of 2021, we also faced significant delays in receiving tissue culture flasks, TranswellsTM, media, pipettes, and other reagents. The delays are approaching a few months and all of the laboratories at Purdue, and at institutions where colleagues work, are encountering them. I fortunately had built up some reserves, but the delays have led to a reduction in the number of experiments we are running and will delay progress until availability returns to normal. This has also led to reduced spending for this year.
Objective 2: Integrate permeability measures with human-based physiological parameters and compound properties relevant to their disposition in the brain to predict Css,brain of compounds.
The proposed timeline is unchanged with the second Objective to be completed in the later project periods.
Future Activities:
Activities for the second project year remain as outlined in the proposal with the modification that some activities of year 1 have been pushed into year 2. These delays have largely been a result of supply issues caused by the SARS-CoV2 pandemic.
Our major goals are to continue on the bidirectional BBB triculture permeability assays with the aim to complete permeability studies by February of 2023. We have begun developing (as outlined in the proposal) the PBPK and QSAR models that incorporate the assays results and characteristics of the compounds tested to date for predicting human exposure
Journal Articles:
No journal articles submitted with this report: View all 1 publications for this projectProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.