Periodic Reporting for period 1 - TOXANOID (Pharmacological safety testing in human adult stem cell-derived organoids)
Período documentado: 2017-04-01 hasta 2018-09-30
Toxicology and pharmacology assays are crucial pillars of modern drug development. They are essential to understand how tissues such as the liver take up and degrade pharmaceutically active compounds. Due to the lack of suitable in vitro systems, these assays are carried out in experimental animals. This practice increases cost and development time but also creates significant ethical issues.
We recently developed a 3D culture system that allows us to expand human tissue stem cells from, amongst others, liver as organoids (mini-organs in a dish) and differentiate them to the respective functional adult tissue in vitro. These stem cell-derived tissues show high cytochrome activity and are able to metabolize drugs in a simple and cost-effective in vitro assay.
With support from the ERC Proof of concept grant “Toxanoid” we aim to prove that our technology can outperform current in vitro systems and replace a significant portion of animal-based toxicology studies. This technological advance would lead to substantial reduction of cost in industrial and academic drug development and overcome practical and ethical pitfalls in animal-based testing.
To this end, our aim was to develop an organoid-based assay to test tissue-drug interactions in three steps: (1) definition of optimal parameters for human in vitro assays to assess gastro-intestinal and hepatic toxicity, (2) Scale-up and (3) Technology transfer and commercial distribution.
Drug metabolizing enzymes are mainly expressed in mature, well-differentiated cell populations. Many organoid types (e.g. liver organoids) are expanded in a stem-cell like state and have to be differentiated to gain full tissue function. Since a high level of differentiation is directly correlated with sensitivity and dynamic range of the assay, we successfully developed a new differentiation protocol that improves hepatocyte fate determination quantitatively and qualitatively. For example, CYP3A4, one of the main drug metabolizing cytochromes, was indistinguishable from primary hepatocytes in expression and activity.
More importantly, the improved differentiation has a strong effect on sensitizing human liver organoids for tissue specific toxic compounds such as Paracetamol. Our improved differentiation protocol significantly increased the sensitivity of differentiated organoids for Acetaminophen (the active substance in Paracetamol) and reduced EC50 values to 3 mM. Indeed, levels of 1-2 mM APAP in circulation are known to cause liver damage in humans.
To test whether drug metabolism in human organoids reflects processing in vivo, we also analyzed the metabolites of Midazolam, Acetaminophen, Tolbutamide and Dextromethorphan by LC-MS. Liver organoids produced the same metabolites as primary hepatocytes, although the rate of metabolite formation was especially for Dextromethorphan slower.
Finally, we tested 12 drugs for toxic effects on differentiated human liver organoids. The assay correctly distinguished toxic from non-toxic compounds and created an almost identical toxicity ranking as primary hepatocytes. Thus, human liver organoids correctly predicted liver toxicity and we proceeded to the scale-up phase.
Since scale-up to 384-well plates reduces the number of organoids per individual well significantly, uniformity of the organoid distribution is paramount to guarantee reproducible results. This applies both to equal seeding per well and a standardized organoid size. Therefore, we tested which organoid number and size-range performed best in toxicity assays. By combining these optimizations, we could generate a reliable 384-well assay that was equivalent to the 48-well assays in sensitivity, accuracy and reproducibility.
The Foundation for Hubrecht organoid technology (HUB) is an organization that acts as interface between academic research at the Hubrecht institute and business partners. HUB has extensive experience with the regulatory requirements and process validation that is necessary to offer organoid assays as commercial product to interested parties. Liver organoid-based drug safety testing in 384-well format has been successfully established at the Hub. The Foundation compiled detailed work-protocols that will be used to transfer the technology to third parties. The Hub currently finishes benchmarking of organoid toxicity assays against various cell lines and primary hepatocytes. After this step, the HUB will approach business partners in the pharmaceutical industry and academia. Several parties have already expressed interest in the technology and will evaluate licensing once our dataset is complete.
We recently developed a 3D culture system that allows us to expand human tissue stem cells from, amongst others, liver as organoids (mini-organs in a dish) and differentiate them to the respective functional adult tissue in vitro. These stem cell-derived tissues show high cytochrome activity and are able to metabolize drugs in a simple and cost-effective in vitro assay.
With support from the ERC Proof of concept grant “Toxanoid” we aim to prove that our technology can outperform current in vitro systems and replace a significant portion of animal-based toxicology studies. This technological advance would lead to substantial reduction of cost in industrial and academic drug development and overcome practical and ethical pitfalls in animal-based testing.
To this end, our aim was to develop an organoid-based assay to test tissue-drug interactions in three steps: (1) definition of optimal parameters for human in vitro assays to assess gastro-intestinal and hepatic toxicity, (2) Scale-up and (3) Technology transfer and commercial distribution.
Drug metabolizing enzymes are mainly expressed in mature, well-differentiated cell populations. Many organoid types (e.g. liver organoids) are expanded in a stem-cell like state and have to be differentiated to gain full tissue function. Since a high level of differentiation is directly correlated with sensitivity and dynamic range of the assay, we successfully developed a new differentiation protocol that improves hepatocyte fate determination quantitatively and qualitatively. For example, CYP3A4, one of the main drug metabolizing cytochromes, was indistinguishable from primary hepatocytes in expression and activity.
More importantly, the improved differentiation has a strong effect on sensitizing human liver organoids for tissue specific toxic compounds such as Paracetamol. Our improved differentiation protocol significantly increased the sensitivity of differentiated organoids for Acetaminophen (the active substance in Paracetamol) and reduced EC50 values to 3 mM. Indeed, levels of 1-2 mM APAP in circulation are known to cause liver damage in humans.
To test whether drug metabolism in human organoids reflects processing in vivo, we also analyzed the metabolites of Midazolam, Acetaminophen, Tolbutamide and Dextromethorphan by LC-MS. Liver organoids produced the same metabolites as primary hepatocytes, although the rate of metabolite formation was especially for Dextromethorphan slower.
Finally, we tested 12 drugs for toxic effects on differentiated human liver organoids. The assay correctly distinguished toxic from non-toxic compounds and created an almost identical toxicity ranking as primary hepatocytes. Thus, human liver organoids correctly predicted liver toxicity and we proceeded to the scale-up phase.
Since scale-up to 384-well plates reduces the number of organoids per individual well significantly, uniformity of the organoid distribution is paramount to guarantee reproducible results. This applies both to equal seeding per well and a standardized organoid size. Therefore, we tested which organoid number and size-range performed best in toxicity assays. By combining these optimizations, we could generate a reliable 384-well assay that was equivalent to the 48-well assays in sensitivity, accuracy and reproducibility.
The Foundation for Hubrecht organoid technology (HUB) is an organization that acts as interface between academic research at the Hubrecht institute and business partners. HUB has extensive experience with the regulatory requirements and process validation that is necessary to offer organoid assays as commercial product to interested parties. Liver organoid-based drug safety testing in 384-well format has been successfully established at the Hub. The Foundation compiled detailed work-protocols that will be used to transfer the technology to third parties. The Hub currently finishes benchmarking of organoid toxicity assays against various cell lines and primary hepatocytes. After this step, the HUB will approach business partners in the pharmaceutical industry and academia. Several parties have already expressed interest in the technology and will evaluate licensing once our dataset is complete.