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Author
Pozo Flores, Marina
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Abstract
The liver plays a central role in drugs metabolization, making it very susceptible to toxicity caused by these agents. Drug induced liver injury (DILI) is an important cause of acute and chronic liver disease. Moreover, it is the most common reason for a drug to fail during development or to be withdrawn from the market. To reduce the attrition rate due to liver toxicity, it needs to be addressed early in the drug discovery process. For that reason, the proposed project will cover the need to develop highly predictive in vitro hepatotoxicity models to be used in early stages of drug discovery.
Human Primary hepatocytes are the gold standard for liver toxicity research; however, they present some limitations such as low availability, limited lifespan, and high interdonor variability. As an alternative, the hepatoma cell line HepG2 is commonly used as a liver model for toxicity studies. HepG2 cells, that derive from a hepatocellular carcinoma, present an unlimited lifespan and express liver specific plasma proteins like albumin.
We hypothesize that HepG2 cells can be used to assess toxicity of compounds early in drug development, to discard highly toxic compounds from the very beginning and reduce attrition rates in clinical phases. Therefore, this work aims to design and validate an in vitro model based on HepG2 cells cultured for a longer period of time in a high-throughput format and following pharmaceutical industry standards (in 384-well plates, 30 μL) to predict hepatoxicity. Under this format, firstly, optimal conditions for HepG2 cell culture were determined. Optimal cell density was established between 250 and 500 cell/well and the maximum culture was set at 5 days (3 days of pretreatment plus 48h post drug addition). Firstly, the model was validated for HTS confirming the reproducibility and robustness of the stablished model. Then, the model was validated using a toolbox of compounds of known toxicity (10 hepatotoxic and 9 hepato-safe compounds). When we compare the obtained results, we conclude that there was not significant difference in treating HepG2 cells the same day of seeding or after 3 days of culture, and that neither of the two approaches showed an ability to accurately predict hepatotoxicity.
As a result, the stablished model is validated for HTS but is not proved to be translacional for the detection of hepatotoxic compounds. As a result, alternative models are suggested to be considered to achieve a better prediction of hepatotoxicity.
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