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High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC
Published on: May 10, 2016
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Cell-based high-throughput screening for the evaluation of reactive metabolite formation potential
Kosuke Harada1, Hiroshi Kohara1, Tomoya Yukawa2
1Drug Safety Research and Evaluation, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan.
Summary
This study introduces a high-throughput glutathione (GSH) consumption assay to predict reactive metabolite (RM) formation. The assay effectively identifies RM potential in early drug discovery, with a co-culture model showing high sensitivity and specificity.
Area of Science:
- Drug Metabolism and Pharmacokinetics
- Toxicology
- Biochemical Assays
Background:
- Reactive metabolites (RMs) are a major cause of drug-induced toxicity.
- Predicting RM formation early in drug discovery is crucial for safety.
- Existing methods for RM assessment can be time-consuming and resource-intensive.
Purpose of the Study:
- To establish and validate a high-throughput in vitro assay system for predicting reactive metabolite formation.
- To optimize assay conditions, including cell type and compound concentration.
- To assess the sensitivity and specificity of the assay for early-stage drug safety evaluation.
Main Methods:
- Development of a glutathione (GSH) consumption assay using HepaRG cells pretreated with D,L-buthionine-(S,R)-sulfoximine (BSO).
- Monitoring of GSH levels as an indicator of RM formation potential.
- Correlation analysis with covalent binding assays.
- Optimization of test compound concentration (100 μM) and evaluation across different cell types (HepG2, PXB-cells, primary hepatocytes) and a co-culture model.
Main Results:
- The GSH consumption assay demonstrated significant decreases in cellular GSH content upon treatment with known RM-forming drugs (ticlopidine, diclofenac) under GSH-reduced conditions.
- A strong correlation (R=0.818) was observed between GSH consumption and covalent binding.
- The assay showed highest sensitivity (56.4%) with HepaRG cells, correlating with high CYP3A4 expression.
- A co-culture model of PXB-cells and HepaRG cells achieved high sensitivity (72.7%) and specificity (85.7%).
Conclusions:
- The developed GSH consumption assay is a reliable and effective method for predicting reactive metabolite formation potential.
- The assay facilitates early-stage risk assessment in drug discovery.
- The optimized co-culture model offers enhanced sensitivity and specificity for RM assessment.
