In a few forthcoming posts, I’d like to share several short articles published by Dr Chris Bohl of Sekisui Xenotech, a technical expert in the field of ADME-Tox tools and applications. I feel they may be of special interest to researchers working in the field of ADME-Tox studies.
Today, we’ll take a look at compounds that exhibit high metabolic stability in hepatocytes and subcellular fractions (S9, microsomes and cytosol), as these can be a challenge for ADME scientists.
Subcellular fraction test systems are easy to source and to use at the bench, and the methods for xenobiotic metabolism in these matrices are largely standardized and accepted by the scientific community. However, these systems are also limited to a 4-6 hour window of time when they are metabolically active and when they can generate quality data.
There have been many interesting advances in the field to address this constraint, however the complexity, amount of labour and cost involved can make these procedures difficult to establish in your lab. Scientists at Sekisui XenoTech have developed and patented the CryostaX® technology, which combines ease of use and cost effectiveness for examining low-turnover compounds in house. Supported by optimized medium, the pooled, plateable human hepatocytes can be cultured for up to 48 hours without a medium change; giving a significantly extended incubation window in which to collect metabolism data (Fig. 1). When compared to alternative methodologies used to assess low-turnover compound metabolism, CryostaX® generates comparable metabolism data in a format that is easier to use and more cost effective. (Fig. 2).
The CryostaX® technology enables Sekisui XenoTech’s scientists to create attaching, customizable pools of hepatocytes that have only undergone a single cryopreservation cycle, versus the two cycles that are required for traditional pooling methods, thereby protecting the cells’ drug metabolizing activities by minimizing the cryoinjury brought on by each round of cryopreservation.
If you have particular needs for your ADME-Tox studies, and would like a special custom pool created by us just for you, then let me know as this can be done at no extra cost. Get in touch via the form below, or contact me directly.
2 Responses
Yes, you can use plateable hepatocytes to help generate clearance data for low-turnover compounds. However, plateable hepatocyte mono-cultures have a tendency to under-predict. If you really want to generate more predictive (in vivo like) data, you would want to use the plateable Cyrostax’s from Xenotech in a more advanced co-culture system like Hurel’s primary hepatic co-cultures. In fact, Astra Zeneca presented a poster at the European ISSX Conference in 2015 which showed significant improvement in clearance prediction using Hurel’s primary hepatic co-cultures with pooled hepatocytes from Xenotech compared to the same lot of hepatocytes in mono-cultures. AZ tested 10 known compounds and 10 proprietary compounds. Using Hurel’s robust primary hepatic co-cultures with the Xenotech cells, they were able to predict clearance values for 18 of the 20 compounds tested, compared to only 15 for the pooled cells in mono-cultures.
Dear Matthew,
Thank you very much for your comment.
Let me address it following a discussion I had with Dr Chris Bohl from Sekisui Xenotech.
CryostaX attaching, pooled primary human hepatocytes (PHH) are one of multiple methodologies available for in vitro DMPK/PK experiments. In the paper that followed the referenced poster, the authors noted that both the CryostaX mono-cultures and the Hurel co-culture were able to predict in vivo hepatic CLint values to within 3-fold of the observed values for greater than 70% of the compounds tested (7/10 and 7/9 respectively); with average fold errors in the predictions of 1.6 for the mono-culture and 2.3 for the co-culture (Bonn et al., 2016) for this particular lot of pooled hepatocytes. It was also stated that the Hurel co-culture “appears somewhat superior regarding overall assay performance, with the opportunity to run incubation up to 72 hours.” These two statement taken together indicated that Cryostax can perform comparably to the co-cultured cell in much shorter experiments, specifically 10 as compared to 72 h. While each method has its own advantages, the CryostaX PHH offer a substantially lower cost, easy to use format that can be utilized on demand when the need arises, and can produce data that is comparable to other methods described in the literature.
Bonn B, Svanberg P, Janefeldt A, Hultman I, and Grime K (2016) Determination of human hepatocyte intrinsic clearance for slowly metabolized compounds: comparison of a primary hepatocyte/stromal cell co-culture with plated primary hepatocytes and HepaRG. Drug Metab Dispos 44: 527-533.
Thanks again for reading my post.
Kind regards,
Isabelle