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Reducing animal experimentation in pre-clinical predictive drug testing by human hepatic in vitro models derived from embryonic stem cells

Final Report Summary - VITROCELLOMICS (Reducing animal experimentation in pre-clinical predictive drug testing by human hepatic in vitro models derived from embryonic stem cells)

The objective of the VITROCELLOMICS project was to establish stable cell lines that reliably reflect human hepatic properties through the development of in vitro models derived from human embryonic stem cells (hESC). The aim has been to deliver such reliable in vitro models that could be used by the pharmaceutical industry to replace the use of animals in investigations on liver toxicity, drug metabolism, uptake and efflux properties of compounds in the drug discovery and development processes. In the pharmaceutical industry, reliable in vitro cell models have the potential to replace current techniques and animal experimentation in the selection and optimisation of lead compounds, and in documentation of a selected drug candidate before it enters clinical phases.

Studies of metabolism and pharmacokinetic properties have become a key activity in the early drug discovery screening programmes. This is mainly driven by the fact that as many as 40 % of new chemical entities were recognised to fail in the late clinical phases because of pharmacokinetic problems. Moreover, adverse drug reactions, most of which are pharmacokinetic-based, are the fourth to sixth leading cause of death in hospitalised patients in the United States (US). Therefore, there is an urgent need for in vitro tools to predict pharmacokinetics and possible toxic reactions of new compounds at an early stage in drug discovery to be able to select high-quality compounds that could be developed into drugs that are safe and easy to administer to the body. Thus, the pharmaceutical companies have made major investments to screen for relevant metabolic properties early in the drug discovery process. A major part of current human-related in vitro methods is based on fractionated tissue of human origin (usually waste material from operations), primary cells, expressed enzymes, hepatoma cell lines, etc. However, a major problem is still the poor predictive power in the available in vitro tools. Today, the pharmaceutical industry still relies heavily on animal models and allometric scaling to predict human pharmacokinetics. Reliable in vitro cell models would replace current techniques and animal experimentation in the selection and optimisation of lead compounds and in documentation of a selected drug candidate before it enters clinical phases. In vitro cell models that could reliably predict human metabolism and disposition would markedly reduce the need for animal experimentation for this purpose.

The overall objectives of the VITROCELLOMICS project were:
- replacing animals in preclinical pharmaceutical development by human hepatic culture systems;
- supporting the predictability of the drug discovery and development process by allowing more reliable and relevant testing in the preclinical phase and preventing weak lead candidates from entering clinical phases through the application of innovative human hepatic cell systems;
- delivering an in vitro testing system with adjacent methodology pertinent for validation in GLP/SOPs environment for absorption, metabolism, and toxicity.
The ultimate aim was to markedly reduce the use of animals in drug testing, refine the model system under consideration, and to replace the animals currently used.

The clinical expertise in the project was manifested by the involvement of two renowned European university hospitals, one SME founded by another well-known European university hospital, and the partnership with one of the leading European pharmaceutical companies. Three partners represent a solid reputed bioengineering background. Other important partners were one SME focused on development of standardised assay conditions for drug testing, and the European Centre for Validation of Alternative Methods (ECVAM).

The protocols used to direct differentiation of hESC lines towards mature hepatic phenotype have been continuously modified, refined, and tested. A milestone resulting of the project was the ability to control the hepatic differentiation via the germ layer definitive endoderm further to functional hepatocytes. Moreover, these derived cells display enzyme activities closer to mature liver than attained with previously derived cells, better clearance properties of drugs, and more distinct expression of biomarkers characteristic for mature liver functionality. Generation of toxicity and drug metabolism data have been completed with the selected test compounds using assays evaluated and selected earlier in the project. Assays used have been optimised for hepatocyte reference cells in the partner laboratories. It was concluded that established toxicity assays are rather poor predictors of clinical toxicity, and that hESC-derived cells may have much higher potential for desired level of predictability. Furthermore, it was demonstrated that the toxicity test method with optical sensing of oxygen consumption (respiration assay) is well suited for incorporation in the project's toxicity test platform. The partners working on test method development used the improved hESC-derived hepatocytes in order to optimise the methods with these cells and to compare toxicity and metabolism with the data from the reference cell types. Respiration was measured in both 96-well Oxoplates and 24-well OxoDishes, which are based on the measurement of luminescence intensity and decay-time, respectively. Both methods are non-invasive and enabled the monitoring of toxicity online, thereby allowing the assessment of the toxicodynamics of the toxic effect of the tested compound.

Respiration in reference cell line (Hep G2) was measured continuously for a desired period of time, and EC50 values were calculated at any chosen time-point. There was a good correlation with other commonly used endpoint assays. All the selected drugs were screened on the reference cell line. It was concluded that Hep G2 cells allow 100 % prediction of parent compound toxicity when used in a multi-assay platform with at least one kinetic assay. Respiration measurements using hESC-derived hepatocytes, cryopreserved primary hepatocytes, and Hep G2 in 24-well respiration assays were also carried out. Diclofenac and amiodarone were tested in a range of concentrations for the determination of the EC50 values. Metabolite balancing and 13C labelling studies were carried out. Using metabolite balancing, a flux map was established for Hep G2 cells.

Now that the VITROCELLOMICS project is finished, the main remaining action is to make the results available for further studies. In addition to the published scientific reports and reports under submission, an invited review article will be published in Toxicology In Vitro that will comprehensively summarises the results. The partners will support AXLR8 and other actions taken to disseminate the VITROCELLOMICS results.

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